Anti-CD38 Antibodies, Anti-CD3 Antibodies, and Bispecific Antibodies, and Uses Thereof

Abstract

The present disclosure relates to anti-CD3 and anti-CD38 antibodies or antigen-binding fragments thereof. The present disclosure also relates to bispecific antibodies that target both CD3 and CD38. To enhance the therapeutic index, the bispecific antibodies may contain a masking domain to minimize systemic toxicity. Unmasking of shielded bispecific antibodies occurs primarily by proteases and enzymes in the tumor microenvironment or diseased tissue. The present disclosure also provides a unique design using human VHO single domain molecules linked to the hinge region of the antibody, which may allow better tissue penetration than conventional antibodies.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 228,195, filed August 2, 2021, the entire contents of which are incorporated herein by reference. Sequence Listing

[0002] This application contains an entire Sequence Listing, which has been submitted electronically in ST.26 format and is hereby incorporated by reference in its entirety. Field of the Disclosure

[0003] The present disclosure relates to biopharmaceuticals, in particular antibodies and bispecific antibodies directed against CD3 and CD38, nucleic acids encoding such antibodies, methods for preparing such antibodies, and methods for the treatment of cancer. [Background technology]

[0004] Background of the Disclosure Antibody-based therapeutics have been successful in treating a variety of diseases, including many cancers. However, improvements to this class of drugs are still needed, especially with regard to increasing their clinical efficacy and safety. One avenue being explored is the genetic engineering of additional and novel antigen-binding sites into antibody-based drugs, such that a single immunoglobulin molecule co-binds two different antigens.

[0005] Also known as cyclic ADP-ribose hydrolase, CD38 is a type II transmembrane glycoprotein with a long C-terminal extracellular domain and a short N-terminal cytoplasmic region (Guedes, Dileepan et al. 2020, Kar, Mehrotra et al. 2020). CD38 is a glycoprotein found on the surface of many immune cells, including CD4+, CD8+, B lymphocytes, and natural killer cells (Orciani, Trubiani et al. 2008, van de Donk and Usmani 2018). More than just a cell type marker, CD38 is an activator of B and T cells. CD38 has multiple functions including extracellular enzyme activity and receptor-mediated regulation of cell adhesion and signal transduction, as shown in Figure 1 (Malavasi, Funaro et al. 1994, Mehta, Shahid et al. 1996, Deaglio, Mehta et al. 2001). CD38 is also a signaling enzyme involved in the metabolism of two novel calcium messenger molecules: CD38 enzymatic activity mediates the synthesis of the calcium releasing second messengers cyclic ADP ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) (Quarona, Zaccarello et al. 2013). Within hematopoietic cells, CD38 can mediate signaling including regulation of lymphocyte proliferation, cytokine release, B and myeloid cell development and survival, and induction of dendritic cell maturation.

[0006] As a receptor, CD38 attaches to CD31 on the surface of T cells, thereby activating those cells to produce various cytokines (Nooka, Kaufman et al. 2019). Also known as PECAM1 (platelet endothelial cell adhesion molecule-1), CD31 is a 130 kDa member of the immunoglobulin superfamily expressed on the surface of circulating platelets, neutrophils, monocytes, and naive B lymphocytes. Functionally, CD31 is thought to function as an adhesion molecule. For example, endothelial cell CD38 binds to CD31 on natural killer cells in order for those cells to adhere to endothelial cells (Glaria and Valledor 2020, Zambello, Barila et al. 2020). CD38 on leukocytes can attach to CD31 on endothelial cells, allowing leukocytes to bind to and migrate through the vessel wall (Quarona, Zaccarello et al. 2013). Such interactions between CD31 and CD38 may play a role in promoting the survival of leukemic cells.

[0007] As a cell surface antigen, CD38 is highly expressed in several hematological malignancies, including multiple myeloma (MM), and has been demonstrated to be a good target for immunotherapy of certain diseases, as shown in Figure 2 (Quarona, Zaccarello et al. 2013). CD38 is upregulated in many cell lines derived from various hematopoietic malignancies, including non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), multiple myeloma (MM), B-cell chronic lymphocytic leukemia (B-CLL), B- and T-cell acute lymphocytic leukemia (ALL), T-cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's lymphoma (HL), and chronic myeloid leukemia (CML). Most primitive multipotent stem cells of the hematopoietic system are also CD38+.

[0008] High expression of CD38 is a diagnostic marker for chronic lymphocytic leukemia (CLL) and is associated with enhanced disease progression (Burgler 2015, Burgler, Gimeno et al. 2015). CD38 is a prognostic factor for acute myeloid leukemia (Zeijlemaker, Grob et al. 2019), chronic lymphocytic leukemia (Damle, Wasil et al. 1999, Malavasi, Deaglio et al. 2011), prostate cancer (Liu, Grogan et al. 2016, Stone 2017), pancreatic cancer (Zhang, Yang et al. 2019), acute B-cell lymphoblastic leukemia (Jiang, Wu et al. 2016), lung cancer (Xu, Chen et al. 2015), hepatocellular carcinoma (Lam, Ng et al. 2019), and triple-negative breast cancer (Yeong, Lim et al. 2018). CD38 is also heterogeneously expressed in colorectal cancer (CRC) (Perenkov, Novikov et al. 2012), independent of tumor localization, tumor grade, or the presence of metastases. CD38 is also a putative functional marker of satellite population cells in human NPC cell lines (Zheng, Liao et al. 2016) and may play an oncogenic role in NPC cells by affecting energy metabolism (Ge, Long et al. 2019).

[0009] Because CD38 expression was high and constant in malignant lymphomas and low in normal lymphoid and myeloid cells and non-hematopoietic tissues, several biologic treatments have been developed (Morandi, Airoldi et al. 2019). In chronic lymphocytic leukemia, CD38 expression is high, and preclinical studies on the use of daratumumab in monotherapy or combination therapy have demonstrated considerable efficacy. CD38 targeting has been used to treat multiple myeloma (MM) and chronic lymphocytic leukemia (CLL). Monoclonal antibodies targeting CD38, such as daratumumab, have shown good therapeutic efficiency in MM, both alone (Usmani, Weiss et al. 2016) and in combination with other standard treatment regimens (Dimopoulos, Oriol et al. 2016, Palumbo, Chanan-Khan et al. 2016). Such therapeutic approaches have further extended the 5-10 year survival rates of MM patients.

[0010] However, many patients eventually relapse due to resistance mechanisms including crystallinity fragment gamma receptor (FcγR)-dependent downregulation of CD38 on tumor cells, as well as inhibition of complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent cellular phagocytosis, as shown in Figure 4 (van de Donk and Usmani 2018). Thus, novel therapeutic approaches are urgently needed.

[0011] Similarly, in other lymphoproliferative disorders, the clinical and clinical data have not been convincing. In these cases, CD38 overexpression can contribute to resistance to checkpoint inhibitors, prompting a number of clinical trials in Hodgkin and non-Hodgkin lymphoma to investigate whether blocking CD38 would enhance the efficacy of checkpoint inhibitors. In the hypoxic tumor microenvironment, NAD +ADP-ribose is released by the salvage pathway and hydrolyzed by CD38 to form ADP-ribose, which is further degraded to AMP via the CD38-CD203a-CD73 pathway. Following this, CD73 dephosphorylates AMP to adenosine. Accumulated extracellular adenosine then binds to receptors on various immune cells and prevents their infiltration and activation. This pathway forms an alternative immunosuppressive mechanism to the PD-1 / PD-L1 pathway, and inhibition of the adenosine pathway has been shown to alleviate immunosuppression in the tumor microenvironment (Ma, Deng et al. 2017, Boison and Yegutkin 2019).

[0012] CD38-NAD + The signaling pathway appears to have a relevant role in the formation of an inhibitory intratumoral microenvironment and promotes the activity of inhibitory cell types such as MDSCs, Tregs, Bregs, and certain subtypes of NK cells. Moreover, it is an important driver of resistance to PD-1 / PD-L1 checkpoint inhibitors. Therefore, CD38 cytotoxic antibodies can exhibit direct tumor activity as well as indirect immunomodulatory antitumor effects. They have been used to treat CD38-positive tumors, specifically MM, with considerable efficacy and a manageable toxicity profile. Substantial presymptomatic evidence supports their use as monotherapy and in combination with certain agents such as BTK inhibitors, as well as CLL. Conversely, other lymphoid malignancies appeared to be less sensitive to anti-CD38 antibodies, with evidence of poor activity of single-agent daratumumab in various types of NHL. Daratumumab and newly developed anti-CD38 antibodies such as isatuximab may find application in combination with standard dosing regimens to enhance cytotoxic responses or with checkpoint inhibitors to overcome acquired resistance. Moreover, anti-CD38 antibodies have not yet been explored in T-cell malignant lymphomas (Calabretta and Carlo-Stella 2020). Thus, there is a need to have more potent and selective CD38 therapeutics available for such indications with better efficacy and safety profiles.

[0013] In prostate cancer, CD38 inhibits cellular NAD + CD38 inhibits tumor metabolism and proliferation by reducing the mitochondrial pool (Chmielewski, Bowlby et al. 2018). CD38 is highly expressed in cervical cancer cells, promotes proliferation, and inhibits apoptosis of cervical cancer cells by affecting mitochondrial function (Liao, Xiao et al. 2014). High expression of CD38 predicted prognosis in patients with esophageal squamous cell carcinoma (Liao, Xiao et al. 2017). CD38 expression or absence showed prognostic value for lung cancer (Karimi-Busheri, Zadorozhny et al. 2011). CD38 knockout suppresses tumor formation in mice and proliferation of human lung cancer cells (Bu, Kato et al. 2018). It was therefore concluded that anti-CD38 therapy may have therapeutic efficacy in lung cancer (Bu, Kato et al. 2018).

[0014] Moreover, CD38, a marker of cell activation in leukemia, myeloma, and solid tumors, has been implicated in HIV infection, type II diabetes, bone metabolism, and several genetically determined conditions (Marlein, Piddock et al. 2019). CD38 overexpression was shown to facilitate CD4 T cell depletion in HIV infection in animal models (Rodriguez-Alba et al. 2019). The enhanced catalytic activity of CD38 may reduce cytoplasmic nicotinamide adenine dinucleotide (NAD) in CD4 T cells and produce a chronic "Warburg effect". In turn, this will reduce mitochondrial function. Meanwhile, ADPR and cADPR, the main catalytic products of CD38, are involved in the regulation of mitochondrial function by their ability to activate calcium channels and upregulate cytoplasmic Ca. 2+ It may increase the concentration of CD38, be transported into the cytoplasm, and even alter mitochondrial integrity. These mechanisms may reduce the viability and regenerative capacity of CD4+ T cells. Thus, shutting down CD38 activity can improve CD4+ T cell activity.

[0015] Overall, as an alternative, the combination of two antibodies (Abs) recognizing different non-overlapping epitopes of CD38 could mediate potent complement-dependent cytotoxicity (CDC), in contrast to Ab monotherapy, which has only weak CDC capacity (Schutze, Petry et al. 2018). Similarly, the combination of one of these Abs recognizing a non-overlapping epitope with daratumumab resulted in dramatically enhanced CDC. Moreover, introducing the E345R HexaBody mutation in the CH3 domain strongly promoted the CDC potency of these Abs against CD38-expressing cells.

[0016] To achieve stronger patient responses, redirected killing of T cells is a desirable mode of action in many therapeutic areas. Various bispecific antibody formats have been shown to mediate T cell redirection in both preclinical and clinical investigations (May, Sapra et al. 2012, Frankel and Baeuerle 2013). These include tandems of scFv fragments and diabody-based formats, with only a few examples of Fc-based bispecific antibody formats reported (Moore, Bautista et al. 2011, May, Sapra et al. 2012, Frankel and Baeuerle 2013). Bispecific formats that include human Fc regions have a longer circulating half-life, which may result in enhanced efficacy and / or reduced frequency of dose regimens. Among the possible Fc-based bispecific formats, one of the preferred formats for redirecting T cell killing is the so-called heavy chain heterodimer format. This format is particularly interesting as it does not allow the aggregation of multiple copies of human CD3 molecules on the T cell surface, thus preventing any T cell inactivation (Klein, Sustmann et al. 2012).

[0017] Bispecific T cell-engaging (BiTE) antibodies belong to a new class of immunotherapeutic agents that recognize, on the one hand, a specific antigen (i.e., tumor antigen) on the surface of target cells and, on the other hand, the CD3ε chain on T lymphocytes (Fumey, Koenigsdorf et al. 2017). By activating T cells through the CD3 complex and recruiting them in close proximity to target cells, BiTE antibodies efficiently induce T cell-mediated cytotoxicity (Brischwein, Schlereth et al. 2006). In MM, bispecific antibodies recognizing B cell maturation antigens or FcRH5 (CD307) have been shown to eliminate tumor plasma cells in preclinical models (Hipp, Tai et al. 2017, Li, Stagg et al. 2017, Seckinger, Delgado et al. 2017). However, FcRH5 expression is restricted to tumor plasma cells, and B cell maturation antigens are abundantly secreted in MM patients (Sanchez, Li et al. 2012). CD38xCD3 bispecific BiTE (Bi38-3) is being tested in clinical trials consisting of two single-chain variable fragments derived from mouse hybridoma cells targeting human CD38 and CD3e (Fayon, Martinez-Cingolani et al. 2021).

[0018] There are two other CD38×CD3 BsAbs for MM and solid tumors, namely AMG424 and GBR1342 (Jiao, Yi et al. 2020). Currently, both of them are based on the structure of Fab Fc(G1)×scFv-Fc(G1) with hetero-Fc domains that lack FcγR receptors and complement binding (Drent, Groen et al. 2016). The modified Fc domain results in the loss of classical Fc-dependent immune effector mechanisms. Antigen-independent cytokine release syndrome (CRS) will occur when the Fc region of the BsAb (which may cause non-specific activation of T cells) binds to FcγR on T cells (Chatenoud, Ferran et al. 1990). These two features may limit the specificity or efficiency of these cognate bispecific antibodies in vivo.

[0019] To prevent off-target toxicity, Fc domain mutations were added to bispecific T cell engagers to improve T cell trafficking and anti-tumor efficacy (Wang, Hoseini et al. 2019). Recently, the anti-CD38 bispecific antibody, AMG424, was shown to eliminate MM cells in preclinical models but trigger “off-tumor” T cell-mediated cytotoxicity against B, T, and NK cells in vitro (Munoz, Mittelbrunn et al. 2008, Zuch de Zafra, Fajardo et al. 2019). Thus, the development of effective and safe bispecific antibodies may contribute to improving the treatment of MM and other cancers. To minimize targeted toxicity, BsAb Fc-mediated immune functions are unfavorable. To avoid or reduce antigen-independent cytokine release syndrome (CRS) resulting from cross-linking of CD3 with Fcγ receptors and subsequent non-specific activation of immune cells, mutations were introduced into the Fc domain to abolish FcγR binding.

[0020] Amgen's CD38xCD3 BsAb also demonstrated a positive correlation between CD3 affinity and cytokine release syndrome (CRS). In Amgen's case, three BsAbs were constructed with the same affinity for CD38 but different dissociation constant Kd values ​​for CD3, with Kd levels of 4.4, 34, and 150-230 nM, respectively. CRS side effects were comparatively studied in cynomolgus monkeys for lead selection. XmAb4 and XmAb5 were not well tolerated in monkeys, as dosing with both mAbs resulted in high levels of CRS effects. Therefore, AMG424, which has a moderate affinity for CD3, was selected for further clinical development, highlighting the importance of the balance between efficacy and safety for these classes of molecules.

[0021] Numerous reports on the limitations of CD3+ T cell redirected antitumor efficacy point to recruitment of non-productive CD3+ T cell subsets, dose-limiting cytokine storm, presence of an immunosuppressive tumor microenvironment (TME), T cell dysfunction and exhaustion due to expression of immune checkpoint molecules, tumor antigen escape, off-target toxicity, and suboptimal efficacy. In May 2021, Pfizer's erlanatamab (BCMA×CD3) was halted in a pivotal clinical trial in MM due to several cases of severe peripheral neuropathy (Biopharmadive.com 2021). In fact, safety, rather than efficacy, is often the primary concern of T cell redirection, as reflected by the relatively limited commercial success of Catumaxomab (voluntary market withdrawal in the US in 2013 and the EU in 2017) and Blinatumomab (global sales of only $379MM USD in 2020, despite regulatory approval since 2014) (Amgen 2021, Wikipedia 2021). Thus, enhancing the therapeutic index of T cell redirection to maximize clinical potential remains an unmet medical need and is highly desirable. Therefore, modulating T cell activation by attenuating CD3-targeting binding affinity while at the same time maintaining antitumor activity is a promising approach to improve the therapeutic window of T cell engager BsAbs.

[0022] Like bispecific T cell engagers, chimeric antigen receptor T cell immunotherapy (CAR-T) directs cytotoxic T cells to malignant cells expressing a specific antigen, and subsequently, these T cells can become activated, proliferate, and release cytokines that lyse tumor cells (Wang, Kaur et al. 2019). With the rapid development of CAR-T technology and the effective outcomes of daratumumab and isatuximab in the clinic, CD38 has become the target of chimeric antigen receptor-redirected T cells (CAR-T cells) for MM (Wu, Zhang et al. 2019). Preclinical data on CD38-CAR-T cells showed significant efficacy in eliminating MM cells in vitro and in vivo, as well as primary malignant cells isolated from patients suffering from MM in vitro. Interestingly, native CD38 expression disappeared after treatment with CD38-CAR-T cells (Drent, Groen et al. 2016). Several studies have revealed that high-affinity CD38-CAR-T cells exert a strong killing effect not only on myeloma cells but also on normal hematopoietic cells expressing CD38 (Chmielewski, Hombach et al. 2004, Drent, Themeli et al. 2017). To reduce the CAR response to non-tumor, CD38-CAR-T cells needed to be optimized for binding affinity (Drent, Themeli et al. 2017, Yu, Yi et al. 2019). The limitations of the use of CD38-specific CAR-T cells may be explained by the expected toxicity of this approach due to the presence of CD38 on normal cells such as NK cells, activated T cells, and B cells. Another limitation is explained by the variable expression of CD38 on cancer cells. Moreover, it is more desirable and convenient to have a drug readily available to increase the availability of T cell therapy.

[0023] While BsAbs made from antibody fragments suffer from biophysical and pharmacokinetic hurdles, a drawback of those constructed in such a format as full-length antibodies is that they multivalently attract co-target antigens in the absence of the first target antigen, leading to non-specific activation and potential toxicity. The present disclosure provides anti-CD38 and anti-CD3 antibodies, including bispecific antibodies directed against CD3 and CD38, to address one or more shortcomings of existing therapies. Summary of the Invention

[0024] Summary of the Disclosure In one aspect, the disclosure provides an anti-CD38 antibody or antigen-binding portion thereof. In some embodiments, the disclosure includes a heavy chain variable region comprising three complementarity determining regions (CDRs) designated HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are, respectively, as follows: SEQ ID NOs:64, 65, and 66; SEQ ID NOs: 64, 83, and 84; SEQ ID NOs: 64, 65, and 85; and SEQ ID NOs: 64, 65, and 86; The present invention provides an anti-CD38 antibody, or antigen-binding portion thereof, selected from the group consisting of:

[0025] In some embodiments, the disclosure includes a light chain variable region comprising three CDRs denoted LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 are, respectively, as follows: SEQ ID NOs:61, 62, and 63; SEQ ID NOs: 76, 77, and 78; SEQ ID NOs: 79, 80, and 81; and SEQ ID NOs: 82, 62, and 78; The present invention provides an anti-CD38 antibody, or antigen-binding portion thereof, selected from the group consisting of:

[0026] In some embodiments, the present disclosure provides an anti-CD38 antibody, or antigen-binding fragment thereof, comprising a heavy chain sequence comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 12-16, or an antigen-binding portion thereof, and a light chain sequence comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 7-11, or an antigen-binding portion thereof.

[0027] In some embodiments, the disclosure provides anti-CD38 antibodies, or antigen-binding fragments thereof, comprising light chain and heavy chain sequences comprising the following, respectively: SEQ ID NOs:2 and 3; SEQ ID NOs:7 and 12; SEQ ID NOs:7 and 13; SEQ ID NOs:7 and 14; SEQ ID NOs:7 and 15; SEQ ID NOs:7 and 16; SEQ ID NOs:8 and 12; SEQ ID NOs:8 and 13; SEQ ID NOs:8 and 14; SEQ ID NOs:8 and 15; SEQ ID NOs:8 and 16; SEQ ID NOs:9 and 12; SEQ ID NOs:9 and 13; SEQ ID NOs:9 and 14; SEQ ID NOs:9 and 15; SEQ ID NOs:9 and 16; SEQ ID NOs:10 and 12; SEQ ID NOs:10 and 13; SEQ ID NOs:10 and 14; SEQ ID NOs:10 and 15; SEQ ID NOs:10 and 16; SEQ ID NOs:11 and 12; SEQ ID NOs:11 and 13; SEQ ID NOs:11 and 14; SEQ ID NOs:11 and 15; or SEQ ID NOs:11 and 16.

[0028] In some embodiments, the disclosure comprises at least one variable heavy chain-only single domain or antigen-binding portion thereof, wherein the at least one variable heavy chain-only single domain has one of the following sequences, respectively: SEQ ID NOs:67, 68, and 69; SEQ ID NOs: 67, 87, and 69; SEQ ID NOs: 67, 88, and 69; SEQ ID NOs: 67, 89, and 69; SEQ ID NOs: 67, 68, and 90; SEQ ID NOs: 70, 91, and 72; SEQ ID NOs: 70, 92, and 72; SEQ ID NOs: 70, 93, and 72; SEQ ID NOs:94, 95, and 96; SEQ ID NOs: 70, 71, and 97; SEQ ID NOs: 73, 98, and 75; SEQ ID NOs: 73, 99, and 75; SEQ ID NOs: 73, 100, and 75; SEQ ID NOs: 73, 101, and 102; and SEQ ID NOs: 73, 103, and 104; The present invention provides an anti-CD38 antibody, or antigen-binding portion thereof, comprising an HCDR1, HCDR2, and HCDR3 selected from:

[0029] In some embodiments, the disclosure provides an anti-CD38 antibody, or antigen-binding portion thereof, comprising at least one variable heavy-only single domain, or antigen-binding portion thereof, wherein the at least one variable heavy-only (VHO) single domain comprises an amino acid sequence, or antigen-binding portion thereof, having at least 85% identity to any one of SEQ ID NOs: 4, and 17-30.

[0030] In another aspect, the disclosure provides an anti-CD3 antibody or antigen-binding portion thereof. In some embodiments, the disclosure includes a heavy chain variable region comprising three complementarity determining regions (CDRs) designated HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are, respectively, as follows: SEQ ID NOs: 105, 108, and 107; SEQ ID NOs: 105, 109, and 107; SEQ ID NOs: 105, 110, and 107; and SEQ ID NOs: 118, 119, and 120, The present invention provides an anti-CD3 antibody, or an antigen-binding portion thereof, selected from the group consisting of:

[0031] In some embodiments, the disclosure includes a light chain variable region comprising three CDRs denoted LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 are, respectively, as follows: SEQ ID NOs: 114, 112, and 113; and SEQ ID NOs: 115, 116, and 117, The present invention provides an anti-CD3 antibody, or an antigen-binding portion thereof, selected from the group consisting of:

[0032] In some embodiments, the disclosure provides an anti-CD3 antibody, or antigen-binding fragment thereof, comprising a heavy chain sequence comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 32-34, and 39, or an antigen-binding portion thereof, and a light chain sequence comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 36-38, or an antigen-binding portion thereof.

[0033] In some embodiments, the disclosure provides anti-CD3 antibodies, or antigen-binding fragments thereof, comprising light chain and heavy chain sequences comprising the following, respectively: SEQ ID NOs: 39 and 38; SEQ ID NOs: 41 and 40; SEQ ID NOs: 31 and 35; SEQ ID NOs: 31 and 36; SEQ ID NOs: 31 and 37; SEQ ID NOs: 32 and 35; SEQ ID NOs: 32 and 36; SEQ ID NOs: 32 and 37; SEQ ID NOs: 33 and 35; SEQ ID NOs: 33 and 36; SEQ ID NOs: 33 and 37; SEQ ID NOs: 34 and 35; SEQ ID NOs: 34 and 36; or SEQ ID NOs: 34 and 37.

[0034] The anti-CD38 or anti-CD3 antibodies, or antigen-binding fragments thereof, disclosed herein may be human, humanized, or chimeric antibodies, or antigen-binding fragments.

[0035] The anti-CD38 or anti-CD3 antibodies, or antigen-binding fragments thereof, disclosed herein may be full-length IgG1, IgG2, IgG3, or IgG4 antibodies, or antigen-binding fragments thereof, such as Fab, F(ab')2, or scFv fragments. The antibody scaffold may be modified to affect functionality, for example to eliminate residual effector functions.

[0036] In another aspect, the present disclosure provides a method for producing a composition comprising the steps of: A first binding arm comprising: a first heavy chain fusion protein comprising, from N-terminus to C-terminus, shield A, protease sequence A, and an IgG heavy chain or an antigen-binding portion thereof; and a first light chain fusion protein comprising, from N-terminus to C-terminus, shield B, protease sequence B, and an IgG light chain or an antigen binding portion thereof; Contains; wherein the IgG heavy chain or antigen-binding portion thereof and the IgG light chain or antigen-binding portion thereof of the first binding arm are capable of targeting the CD3-related pathway and comprise an anti-CD3 antibody or antigen-binding fragment described herein; and A second binding arm comprising: a second heavy chain fusion protein comprising, from N-terminus to C-terminus, a shield C, a protease sequence C, and an IgG heavy chain or an antigen-binding portion thereof; and a second light chain fusion protein comprising, from N-terminus to C-terminus, a shield D, a protease sequence D, and an IgG light chain or an antigen-binding portion thereof; Contains; wherein the IgG heavy chain or antigen-binding portion thereof and the IgG light chain or antigen-binding portion thereof of the second binding arm are capable of targeting the CD38-associated pathway and comprise an anti-CD38 antibody or antigen-binding fragment as described herein. The present invention provides a bispecific antibody comprising: Shields A-D can be the same or different from one another, and protease sequences A-D can be the same or different from one another. Shields A-D and protease sequences A-D are optional, meaning that some or all of them can be present or absent.

[0037] In certain embodiments, the present disclosure provides a method for the preparation of a method for treating a pulmonary artery disease comprising the steps of: A first binding arm comprising: a first heavy chain fusion protein comprising, from N-terminus to C-terminus, signal sequence A-shield A-linker A-protease sequence A-linker B-IgG heavy chain or antigen binding portion thereof; and A first light chain fusion protein comprising from N-terminus to C-terminus: signal sequence B-shield B-linker C-protease sequence B-linker D-IgG light chain or an antigen binding portion thereof; Contains; wherein the IgG heavy chain or antigen-binding portion thereof and the IgG light chain or antigen-binding portion thereof of the first binding arm are capable of targeting the CD3-related pathway and comprise an anti-CD3 antibody or antigen-binding fragment described herein; and A second binding arm comprising: A second heavy chain fusion protein comprising, from N-terminus to C-terminus, a signal sequence C-shield C-linker E-protease sequence C-linker F-IgG heavy chain or an antigen-binding portion thereof; and A second light chain fusion protein comprising from N-terminus to C-terminus: signal sequence, D-shield, D-linker, G-protease sequence, D-linker, H-IgG light chain, or an antigen-binding portion thereof; Contains; wherein the IgG heavy chain or antigen-binding portion thereof and the IgG light chain or antigen-binding portion thereof of the second binding arm are capable of targeting the CD38-associated pathway and comprise an anti-CD38 antibody or antigen-binding fragment as described herein. The present invention provides a bispecific antibody comprising: The signal sequences A-D can be the same or different from each other, and the linkers A-H can be the same or different from each other. The signal sequences A-D, shields A-D, protease sequences A-D, and linkers A-H are optional, meaning that some or all of them can be present or absent.

[0038] In another aspect, the present disclosure provides a method for producing a composition comprising the steps of: A first binding arm comprising: a first heavy chain fusion protein comprising, from N-terminus to C-terminus, shield A, protease sequence A, and an IgG heavy chain or an antigen-binding portion thereof; and a first light chain fusion protein comprising, from N-terminus to C-terminus, shield B, protease sequence B, and an IgG light chain or an antigen binding portion thereof; Contains; wherein the IgG heavy chain or antigen-binding portion thereof and the IgG light chain or antigen-binding portion thereof of the first binding arm are capable of targeting the CD3-related pathway and comprise an anti-CD3 antibody or antigen-binding fragment described herein; and A second binding arm comprising: a second heavy chain fusion protein comprising, from N-terminus to C-terminus, a shield C, a protease sequence C, and an IgG heavy chain (comprising at least one variable heavy chain only (VHO) single domain or antigen binding portion thereof); Contains; wherein at least one single domain of only the variable heavy chain or an antigen-binding portion thereof is capable of targeting the CD38-related pathway and comprises an anti-CD38 VHO or antigen-binding fragment as described herein. The present invention provides a bispecific antibody comprising: Shields A-C can be the same or different from one another, and protease sequences A-C can be the same or different from one another. Shields A-C and protease sequences A-C are optional, meaning that some or all of them can be present or absent.

[0039] In certain embodiments, the present disclosure provides a method for the preparation of a method for treating a pulmonary artery disease comprising the steps of: A first binding arm comprising: a first heavy chain fusion protein comprising, from N-terminus to C-terminus, signal sequence A-shield A-linker A-protease sequence A-linker B-IgG heavy chain or antigen binding portion thereof; and A first light chain fusion protein comprising from N-terminus to C-terminus: signal sequence B-shield B-linker C-protease sequence B-linker D-IgG light chain or an antigen binding portion thereof; Contains; wherein the IgG heavy chain or antigen-binding portion thereof and the IgG light chain or antigen-binding portion thereof of the first binding arm are capable of targeting the CD3-related pathway; and A second binding arm comprising: A second heavy chain fusion protein comprising, from N-terminus to C-terminus, a signal sequence C-shield C-linker E-protease sequence C-linker F-IgG heavy chain (comprising at least one single domain of only the variable heavy chain or an antigen binding portion thereof); Contains; wherein at least one single domain of only the variable heavy chain or an antigen-binding portion thereof is capable of targeting the CD38-related pathway and comprises an anti-CD38 VHO or antigen-binding fragment as described herein. The present invention provides a bispecific antibody comprising: The signal sequences A-C can be the same or different from each other, and the linkers A-F can be the same or different from each other. The signal sequences A-C, shields A-C, protease sequences A-C, and linkers A-F are optional, meaning that some or all of them can be present or absent.

[0040] In certain embodiments, in the bispecific antibodies disclosed herein, the IgG is human IgG1, IgG2, IgG3, or IgG4.

[0041] In certain embodiments, in the bispecific antibodies disclosed herein, the first binding arm is monovalent and the second binding arm is monovalent, bivalent, or multivalent.

[0042] In certain embodiments, in the bispecific antibodies disclosed herein, the second binding arm comprises, in tandem, two or three IgG variable heavy chain-only single domains, optionally connected via one or more linker sequences.

[0043] In certain embodiments, in the bispecific antibodies disclosed herein, the first binding arm (the anti-CD3 arm) comprises one of the following: a heavy chain variable region comprising three complementarity determining regions (CDRs) designated HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are selected from SEQ ID NOs: 105, 108, and 107; SEQ ID NOs: 105, 109, and 107; SEQ ID NOs: 105, 110, and 107; and SEQ ID NOs: 118, 119, and 120; and a light chain variable region, comprising three CDRs denoted LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 are selected from: SEQ ID NOs: 114, 112, and 113; and SEQ ID NOs: 115, 116, and 117, respectively; Includes.

[0044] In certain embodiments, in the bispecific antibodies disclosed herein, the second binding arm (the anti-CD38 arm) comprises the following: a heavy chain variable region comprising three complementarity determining regions (CDRs) denoted HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are selected from: SEQ ID NOs: 64, 65, and 66; SEQ ID NOs: 64, 83, and 84; SEQ ID NOs: 64, 65, and 85; and SEQ ID NOs: 64, 65, and 86; and a light chain variable region comprising three CDRs denoted LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 are selected from: SEQ ID NOs: 61, 62, and 63; SEQ ID NOs: 76, 77, and 78; SEQ ID NOs: 79, 80, and 81; and SEQ ID NOs: 82, 62, and 78, respectively; Includes.

[0045] In certain embodiments, in the bispecific antibodies disclosed herein, the second binding arm (anti-CD38 arm) comprises at least one variable heavy chain-only single domain or antigen-binding portion thereof, wherein said at least one variable heavy chain-only single domain is selected from the group consisting of SEQ ID NOs: 67, 68, and 69; SEQ ID NOs: 67, 87, and 69; SEQ ID NOs: 67, 88, and 69; SEQ ID NOs: 67, 89, and 69; SEQ ID NOs: 67, 68, and 90; It comprises three CDRs (HCDR1, HCDR2, and HCDR3) selected from SEQ ID NOs: 70, 91, and 72; SEQ ID NOs: 70, 92, and 72; SEQ ID NOs: 70, 93, and 72; SEQ ID NOs: 94, 95, and 96; SEQ ID NOs: 70, 71, and 97; SEQ ID NOs: 73, 98, and 75; SEQ ID NOs: 73, 99, and 75; SEQ ID NOs: 73, 100, and 75; SEQ ID NOs: 73, 101, and 102; and SEQ ID NOs: 73, 103, and 104.

[0046] In a specific embodiment, in the bispecific antibody disclosed herein, the IgG heavy chain of the first binding arm (anti-CD3 arm) comprises an amino acid sequence selected from SEQ ID NOs: 31 to 34, 39, and 41, an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 31 to 34, 39, and 41, or an antigen-binding portion thereof, and the IgG light chain of the first binding arm comprises an amino acid sequence selected from SEQ ID NOs: 35 to 38 and 40, an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 35 to 38 and 40, or an antigen-binding portion thereof.

[0047] In a specific embodiment, in the bispecific antibody disclosed herein, the IgG heavy chain of the second binding arm (anti-CD38 arm) comprises an amino acid sequence selected from SEQ ID NOs: 3 to 6 and 12 to 30, an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 3 to 6 and 12 to 30, or an antigen-binding portion thereof, and the IgG light chain of the second binding arm comprises an amino acid sequence selected from SEQ ID NOs: 2 and 7 to 11, an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 2 and 7 to 11, or an antigen-binding portion thereof.

[0048] In certain embodiments, in the bispecific antibodies disclosed herein, at least one single domain of only the variable heavy chain comprises an amino acid sequence selected from SEQ ID NOs: 4-6 and 17-30, an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 4-6 and 17-30, or an antigen-binding portion thereof.

[0049] In certain embodiments, in the bispecific antibodies disclosed herein, shield A, shield B, shield C, and shield D are each independently selected from the amino acid sequences set forth in SEQ ID NOs: 42-52.

[0050] In certain embodiments, in the bispecific antibodies disclosed herein, protease sequence A, protease sequence B, protease sequence C, and protease sequence D are each independently selected from the amino acid sequences set forth in SEQ ID NOs: 53 to 60.

[0051] In certain embodiments, in the bispecific antibodies disclosed herein, the IgG heavy chain and the IgG light chain of the first binding arm (anti-CD3 arm) comprise the amino acid sequences set forth in SEQ ID NOs: 39 and 38; SEQ ID NOs: 41 and 40; SEQ ID NOs: 31 and 35; SEQ ID NOs: 31 and 36; SEQ ID NOs: 31 and 37; SEQ ID NOs: 32 and 35; SEQ ID NOs: 32 and 36; SEQ ID NOs: 32 and 37; SEQ ID NOs: 33 and 35; SEQ ID NOs: 33 and 36; SEQ ID NOs: 33 and 37; SEQ ID NOs: 34 and 35; SEQ ID NOs: 34 and 36; or SEQ ID NOs: 34 and 37, respectively.

[0052] In certain embodiments, in the bispecific antibodies disclosed herein, the IgG light chain and the IgG heavy chain of the second binding arm (anti-CD38 arm) comprise the amino sequences set forth in SEQ ID NOs:2 and 3; SEQ ID NOs:7 and 12; SEQ ID NOs:7 and 13; SEQ ID NOs:7 and 14; SEQ ID NOs:7 and 15; SEQ ID NOs:7 and 16; SEQ ID NOs:8 and 12; SEQ ID NOs:8 and 13; SEQ ID NOs:8 and 14; SEQ ID NOs:8 and 15; SEQ ID NOs:8 and 16; SEQ ID NOs:9 and 12; SEQ ID NOs:9 and 13; SEQ ID NOs:9 and 14; SEQ ID NOs:9 and 15; SEQ ID NOs:9 and 16; SEQ ID NOs:10 and 12; SEQ ID NOs:10 and 13; SEQ ID NOs:10 and 14; SEQ ID NOs:10 and 15; SEQ ID NOs:10 and 16; SEQ ID NOs:11 and 12; SEQ ID NOs:11 and 13; SEQ ID NOs:11 and 14; SEQ ID NOs:11 and 15; or SEQ ID NOs:11 and 16, respectively.

[0053] In certain embodiments, in the bispecific antibodies disclosed herein, at least one variable heavy chain-only single domain comprises two variable heavy chain-only single domains each independently selected from SEQ ID NOs: 4-6 and 17-30.

[0054] In certain embodiments of the bispecific antibodies disclosed herein, at least one variable heavy chain only single domain comprises, from N-terminus to C-terminus, SEQ ID NO:4 and SEQ ID NO:5, optionally connected via a linker; SEQ ID NO:4 and SEQ ID NO:6, optionally connected via a linker; SEQ ID NO:5 and SEQ ID NO:6, optionally connected via a linker; SEQ ID NO:5 and SEQ ID NO:4, optionally connected via a linker; SEQ ID NO:6 and SEQ ID NO:5, optionally connected via a linker; or SEQ ID NO:6 and SEQ ID NO:4, optionally connected via a linker.

[0055] In certain embodiments, the antibodies or bispecific antibodies disclosed herein comprise a modified Fc to extend the half-life of the bispecific antibody, reduce effector functionality of the bispecific antibody, increase resistance of the bispecific antibody to proteolysis, facilitate manufacturing of the bispecific antibody by Fc heterodimerization, facilitate multimerization of the bispecific antibody, and / or improve manufacturing and drug stability of the bispecific antibody.

[0056] In another aspect, the disclosure provides a conjugate comprising an antibody or bispecific antibody disclosed herein linked to a moiety, such as a cytotoxic agent.

[0057] In another aspect, the present disclosure provides a composition comprising an antibody or bispecific antibody disclosed herein, or a conjugate disclosed herein. In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an antibody or bispecific antibody disclosed herein, or a conjugate disclosed herein, and a pharma- ceutically acceptable carrier.

[0058] In another aspect, the disclosure provides a nucleic acid encoding an anti-CD38 antibody, or an antigen-binding portion thereof.

[0059] In another aspect, the disclosure provides a nucleic acid encoding an anti-CD3 antibody, or an antigen-binding portion thereof.

[0060] In another aspect, the disclosure provides a nucleic acid encoding a bispecific antibody, a first heavy chain fusion protein, a first light chain fusion protein, a second heavy chain fusion protein, or a second light chain fusion protein disclosed herein.

[0061] In another aspect, the disclosure provides a recombinant vector, such as an expression vector, comprising a nucleic acid disclosed herein.

[0062] In another aspect, the disclosure provides a host cell comprising a recombinant vector, such as an expression vector, or a nucleic acid disclosed herein.

[0063] In another aspect, the disclosure provides a method of preparing an antibody or bispecific antibody disclosed herein comprising culturing a host cell disclosed herein, growing the host cell in a host cell culture, providing host cell culture conditions in which a nucleic acid disclosed herein is expressed, and recovering the antibody or bispecific antibody from the host cell or host cell culture. In certain embodiments, the bispecific antibody is obtained using controlled Fab arm exchange.

[0064] In another aspect, the disclosure provides a method of treating or preventing a CD38-mediated disease or disorder in a subject in need thereof, comprising administering to the subject a medicamentically effective amount of an antibody, bispecific antibody, conjugate, or pharmaceutical composition disclosed herein. In certain embodiments, the disease or disorder is selected from human cancer, including gastric and colorectal cancer, pancreatic cancer, prostate cancer, lung cancer, hepatocellular carcinoma, triple-negative breast cancer, nasopharyngeal cancer, cervical cancer, hematological malignancies (such as MM, lymphoma, acute myeloid leukemia, chronic lymphocytic leukemia, acute B lymphoblastic leukemia, and others), heart disease, viral infections, including HIV infection, asthma and other respiratory inflammatory diseases, allergic airway diseases, maternal tolerance, autism spectrum disorders, glomerulosclerosis, inflammatory bowel disease, rheumatoid arthritis, diabetes mellitus, chronic autoimmune thyroiditis, and neurodegenerative and neuroinflammatory diseases, such as Graves' disease, Alzheimer's disease. In some embodiments, the CD38-mediated disease or disorder is selected from human cancers, including gastric and colorectal cancer, pancreatic cancer, prostate cancer, lung cancer, hepatocellular carcinoma, triple-negative breast cancer, nasopharyngeal cancer, cervical cancer, and hematological malignancies.

[0065] In another aspect, the disclosure provides a method of mediating CD38 in a subject in need thereof, comprising administering to the subject an effective amount of an antibody, bispecific antibody, or pharmaceutical composition disclosed herein.

[0066] This and other embodiments of the present disclosure are described in greater detail herein. [Brief description of the drawings]

[0067] [Figure 1A-B] Figure 1 is a schematic diagram illustrating the role of CD38 in immune cells. Figure 1A shows that CD38 is primarily expressed in immune cells and metabolizes nicotinamide nucleotides (NAD+ and NMN) to ADPR and cADPR (Hogan, Chini et al. 2019). Figure 1B shows multiple immune suppressive mechanisms that impede antitumor immunity. [Diagram 2] FIG. 2 shows the mRNA expression profile of CD38. [Diagram 3] FIG. 3 illustrates the mechanism of action of anti-CD38 antibodies. [Figure 4] FIG. 4 illustrates the mechanisms of resistance to anti-CD38 antibodies. [Figure 5A-B] Figure 5 is a schematic diagram of a CD38xCD3 bispecific antibody. Figure 5A shows a shielded bispecific antibody comprising two different sets of heavy chain (HC) and light chain (LC) combinations, indicated by the annotations "first arm" and "second arm". Both arms can be shielded. Figure 5B shows a bispecific antibody comprising two different sets of heavy chain (HC) and light chain (LC) combinations without a shielding or masking domain. [Figure 5C-D] Figure 5C illustrates a particular component consisting of a CD3 arm (first arm) open reading frame and a CD38 binding arm (second arm) open reading frame with shielding, and Figure 5D illustrates a particular component consisting of a CD3 arm (first arm) open reading frame and a CD38 binding arm (second arm) open reading frame without shielding. [Figure 6A-B]Figure 6 is a schematic diagram of a CD3xCD38 bispecific antibody comprising one or more anti-CD38 variable heavy chain only (VHO) single domains. Figure 6A shows two examples of such a CD3xCD38 bispecific antibody: a bispecific antibody comprising a single VHO in the "second arm", the arm targeting CD38 (left panel); a bispecific antibody comprising a CD38 binding arm (second arm) comprising two VHOs fused together (right panel). In both examples, the VHOs are connected to a masking domain. Figure 6B shows in the left panel a bispecific antibody comprising a single VHO in the "second arm", the arm targeting CD38; and in the right panel a bispecific antibody comprising a CD38 binding arm (second arm) comprising two VHOs fused together. In both examples, the VHOs are not connected to a masking domain. [Figure 6C-D] Figure 6C illustrates a particular component consisting of a CD3 arm (first arm) open reading frame and a CD38 binding arm (second arm) open reading frame with shielding, and Figure 6D illustrates a particular component consisting of a CD3 arm (first arm) open reading frame and a CD38 binding arm (second arm) open reading frame without shielding. [Figure 7] FIG. 7 is a schematic representation of the protease digestive removal of the masking domain from a CD38×CD3 bispecific antibody. [Figure 8]Figure 8 demonstrates concentration-dependent ELISA binding of anti-CD3 antibodies to recombinant human (Figure 8A) and cynomolgus monkey (Figure 8B) CD3 delta and epsilon domain proteins in an ELISA assay. A positive control anti-CD3 antibody, SP34, was used in the binding assay. SP34 has the heavy chain variable domain sequence: EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS (SEQ ID NO: 127) and the light chain variable domain sequence: QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL (SEQ ID NO: 38). The y-axis is the binding response expressed as optical density in absorbance units at 450 nm. The x-axis is the concentration of molecules tested. SP34 and SP34 v8 (SEQ ID NO: 38 as the light chain and SEQ ID NO: 39 as the heavy chain) had strong binding to human and cyno delta and epsilon domain proteins. [Figure 9] Figure 9 demonstrates concentration-dependent flow cytometry binding of anti-CD3 antibodies to CD3-bearing primary human T cells from a PBMC preparation. The y-axis is the binding response expressed as global mean fluorescence intensity (gMFI from AlexaFluor647 detection). The x-axis is the concentration of antibody evaluated. [Figure 10A-C]FIG. 10 demonstrated concentration-dependent ELISA binding of anti-CD38 antibodies to (FIG. 10A, D, and E) recombinant human, (FIG. 10B) recombinant cynomolgus, and (FIG. 10C) mouse recombinant CD38 proteins. Concentration-dependent ELISA binding of anti-CD38 single domain antibodies to human CD38 is shown in FIG. 10D and E. The y-axis was the binding response expressed as optical density in absorbance units at 450 nm. The x-axis was the concentration of the tested molecule. The results showed that molecules with SEQ ID NOs: 2 and 3 (mAb) and SEQ ID NOs: 4, 5, 6, 17, 18, 20, 21, 24, and 29 (VHO), respectively, bind to recombinant human CD38 protein. Molecules with SEQ ID NOs: 4 and 5, respectively, may also bind to recombinant cynomolgus CD38 protein. Molecules containing SEQ ID NO: 4 may also bind to recombinant mouse CD38 protein. [Fig. 10D-E] FIG. 10 demonstrated concentration-dependent ELISA binding of anti-CD38 antibodies to (FIG. 10A, D, and E) recombinant human, (FIG. 10B) recombinant cynomolgus, and (FIG. 10C) mouse recombinant CD38 proteins. Concentration-dependent ELISA binding of anti-CD38 single domain antibodies to human CD38 is shown in FIG. 10D and E. The y-axis was the binding response expressed as optical density in absorbance units at 450 nm. The x-axis was the concentration of the tested molecule. The results showed that molecules with SEQ ID NOs: 2 and 3 (mAb) and SEQ ID NOs: 4, 5, 6, 17, 18, 20, 21, 24, and 29 (VHO), respectively, bind to recombinant human CD38 protein. Molecules with SEQ ID NOs: 4 and 5, respectively, may also bind to recombinant cynomolgus CD38 protein. Molecules containing SEQ ID NO: 4 may also bind to recombinant mouse CD38 protein. [Figure 11]Figure 11 demonstrates the dose response of CD38xCD3 bispecific antibody T cell activation from a series of CD3-bearing Jurkat T cell reporter assays in the presence of CD38-bearing H929 multiple myeloma cells. CD38 VHO with SEQ ID NO: 4 was combined with different CD3 arms to create the indicated CD38xCD3 bispecific antibodies (defined as SEQ ID NO: 4xCris7 v4 (heavy chain variable sequence with SEQ ID NO: 33, light chain variable sequence with SEQ ID NO: 36), SEQ ID NO: 4xCris7 v3 (heavy chain variable sequence with SEQ ID NO: 32, light chain variable sequence with SEQ ID NO: 37), and SEQ ID NO: 4xSP34 v8 (heavy chain variable sequence with SEQ ID NO: 39, light chain variable sequence with SEQ ID NO: 38), respectively). The y-axis is the reporter assay response in relative light units. The x-axis is the concentration of the tested molecule. [Figure 12] Figure 12 demonstrates the dose response of CD38xCD3 bispecific antibody T cell activation from a series of CD3-bearing Jurkat T cell reporter assays in the presence of CD38-bearing L363 multiple myeloma cells. CD38 VHO with SEQ ID NO: 4 was combined with different CD3 arms to create the indicated CD38xCD3 bispecific antibodies (defined as SEQ ID NO: 4xCris7 v3 (heavy chain variable sequence with SEQ ID NO: 32, light chain variable sequence with SEQ ID NO: 37), SEQ ID NO: 4xCris7 v4 (heavy chain variable sequence with SEQ ID NO: 33, light chain variable sequence with SEQ ID NO: 36), and SEQ ID NO: 4xSP34, respectively). The y-axis is the reporter assay response in relative light units. The x-axis is the concentration of the tested molecule. [Figure 13] Figure 13 demonstrates the dose response of CD38xCD3 bispecific antibody T cell activation from a series of CD3-bearing Jurkat T cell reporter assays in the presence of CD38-bearing RPMI 8226 multiple myeloma cells. CD38 VHO with SEQ ID NO: 4 was combined with different CD3 arms to create the indicated CD38xCD3 bispecific antibodies. A strong T cell activation response was observed. The y-axis is the reporter assay response in relative light units. The x-axis is the concentration of the tested molecule. [Figure 14A-B]Figure 14 demonstrates the dose response of CD38xCD3 bispecific antibodies directing primary human T cell killing of H929, a CD38-bearing multiple myeloma cell line. Two different lots of PBMCs (Figures 14A and 14B) were used. CD38 VHO with SEQ ID NO: 4 was combined with different CD3 arms to create the CD38xCD3 bispecific antibodies shown in Figure 14. A strong T cell killing response was observed. The y-axis is the percent cell killing. The x-axis is the concentration of the molecules evaluated. [Figure 15A-B] Figure 15 demonstrates the dose response of CD38xCD3 bispecific antibodies directing primary human T cell killing of RPMI 8226, a CD38-bearing multiple myeloma cell line. Four different lots of PBMCs (Figures 15A, 15B, 15C, and 15D) were used. CD38 VHO with SEQ ID NO: 4 was combined with different CD3 arms to create the CD38xCD3 bispecific antibodies shown in Figure 15. A strong T cell killing response was observed. The y-axis is the percent cell killing. The x-axis is the concentration of the molecules evaluated. [Fig. 15C-D] Figure 15 demonstrates the dose response of CD38xCD3 bispecific antibodies directing primary human T cell killing of RPMI 8226, a CD38-bearing multiple myeloma cell line. Four different lots of PBMCs (Figures 15A, 15B, 15C, and 15D) were used. CD38 VHO with SEQ ID NO: 4 was combined with different CD3 arms to create the CD38xCD3 bispecific antibodies shown in Figure 15. A strong T cell killing response was observed. The y-axis is the percent cell killing. The x-axis is the concentration of the molecules evaluated. [Figure 16A-B] Figure 16 demonstrates the dose response of CD38xCD3 bispecific antibodies directing primary human T cell killing of L363, a CD38-bearing multiple myeloma cell line. Different lots of PBMCs (Figures 16A, 16B, 16C, and 16D) were used. CD38 VHO with SEQ ID NO: 4 was combined with different CD3 arms to create the CD38xCD3 bispecific antibodies shown in Figure 16. A strong T cell killing response was observed. The y-axis is the percent cell killing. The x-axis is the concentration of the molecules evaluated. [Fig. 16C-D]Figure 16 demonstrates the dose response of CD38xCD3 bispecific antibodies directing primary human T cell killing of L363, a CD38-bearing multiple myeloma cell line. Different lots of PBMCs (Figures 16A, 16B, 16C, and 16D) were used. CD38 VHO with SEQ ID NO: 4 was combined with different CD3 arms to create the CD38xCD3 bispecific antibodies shown in Figure 16. A strong T cell killing response was observed. The y-axis is the percent cell killing. The x-axis is the concentration of the molecules evaluated. [Figure 17A] FIG. 17 shows a mouse model of efficacy of two CD38×CD3 bispecific antibodies defined as SEQ ID NO:4×SP34 v8 (comprising CD38 SEQ ID NO:4 and CD3 SP34 v8 (SEQ ID NO:38 as the light chain and SEQ ID NO:39 as the heavy chain); and SEQ ID NO:4×40G5 (comprising CD38 SEQ ID NO:4 and 40G5 (SEQ ID NO:40 as the light chain and SEQ ID NO:41 as the heavy chain). FIG. 17A shows two study designs, (“Donor 1 / 2” and “Donor 3 / ”), of the mouse efficacy model using transplanted human PBMCs and human NCI-H929 multiple myeloma cells treated with bispecific antibodies comprising novel anti-CD38 specificities and a positive control anti-CD3 antibody with known anti-CD3 bispecific antibody activity. [Fig. 17B-C] Figures 17B-17C (corresponding to study design donor 1 / 2) and Figures 17D-17E (corresponding to study design donor 3 / 4) show tumor growth inhibition of human NCI-H929 multiple myeloma cells in a mouse efficacy model using transplanted human PBMCs and treated with the antibodies shown in Figures 17B-17E. [Fig. 17D-E] Figures 17B-17C (corresponding to study design donor 1 / 2) and Figures 17D-17E (corresponding to study design donor 3 / 4) show tumor growth inhibition of human NCI-H929 multiple myeloma cells in a mouse efficacy model using transplanted human PBMCs and treated with the antibodies shown in Figures 17B-17E. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] Detailed Description of the Disclosure definition All publications, including but not limited to patents and patent applications, cited herein are hereby incorporated by reference as if fully set forth. In the event that the specific content of a publication cited herein contradicts or is contrary to the present disclosure, the present disclosure controls.

[0069] Any embodiment of the disclosure described herein, including those described only in a section of the specification describing specific aspects of the disclosure and those described only in the examples or drawings, may be combined with any other one or more embodiments, unless expressly disclaimed or inappropriate.

[0070] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0071] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, exemplary materials and methods are described herein. In describing and claiming the present disclosure, the following terminology is used.

[0072] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, etc.

[0073] "Antibody" is intended in a broad sense to include immunoglobulin molecules including monoclonal antibodies, including murine, human, humanized and chimeric monoclonal antibodies, antibody fragments, bispecific or multispecific antibodies, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies, and any other modified configuration of immunoglobulin molecule that contains an antigen binding site of the required specificity.

[0074] A "full-length antibody molecule" is composed of two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (composed of domains CH1, hinge, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into regions of hypervariability, named complementarity determining regions (CDRs), interspersed with framework regions (FRs). Each VH and VL is composed of three CDRs and four FR segments, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0075] "Complementarity determining regions (CDRs)" are the "antigen-binding sites" in an antibody. CDRs can be defined using various terms: (i) three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3), Complementarity determining regions (CDRs) are based on sequence variability (Wu and Kabat 1970) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). (ii) three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). "Hypervariable region", "HVR" or "HV" refers to the region of an antibody variable domain that is structurally hypervariable, as defined by Chothia and Lesk (Chothia and Lesk 1987). The International Immunogenetics (IMGT) database (http: / / www_imgt_org) provides standardized numbering and definition of antigen-binding sites. The correspondence between CDR, HV and IMGT delineations has been described (Lefranc, Pommie et al. 2003). The terms "CDR", "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2" and "LCDR3" as used herein include CDRs defined by any of the above methods, Kabat, Chothia or IMGT, unless otherwise expressly stated herein.

[0076] Immunoglobulins can be assigned to five major classes, IgA, IgD, IgE, IgG, and IgM, depending on the heavy chain constant region amino acid sequence. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, kappa (κ) and lambda (λ), based on the amino acid sequence of their constant region.

[0077] An "antibody fragment", "antigen-binding fragment", or "antigen-binding portion" refers to a portion of an immunoglobulin molecule that retains a heavy and / or light chain antigen-binding site, such as heavy chain complementarity determining regions (HCDRs) 1, 2 and 3, light chain complementarity determining regions (LCDRs) 1, 2 and 3, a heavy chain variable region (VH), or a light chain variable region (VL). Antibody fragments include the well-known Fab, F(ab')2, Fd and Fv fragments, as well as domain antibodies (dAbs) consisting of a single VH domain. The VH and VL domains can be linked together via a synthetic linker to form various types of single chain antibody designs in which the VH / VL domains can pair intramolecularly or intermolecularly, for example, as described in International Disclosure Publication Nos. WO 1998 / 44001, WO 1988 / 01649, WO 1994 / 13804 and WO 1992 / 01047, where the VH and VL domains are expressed as separate single chain antibody constructs to form a monovalent antigen binding site, such as a single chain Fv (scFv) or diabody.

[0078] "Antibody mimic" refers to an engineered antibody protein that exhibits specific binding to a target. For example, the antibody mimic can be an Affibody, a DARPin, an Anticalin, an Avimer, a Versa body, or a Duocalin.

[0079] "Monoclonal antibody" refers to an antibody population with a single amino acid composition in each heavy and light chain, except for possible known modifications such as removal of the C-terminal lysine from the antibody heavy chain. A monoclonal antibody typically binds to one antigen epitope, except for example, a bispecific monoclonal antibody binds to two distinct antigen epitopes. A monoclonal antibody can have heterogeneous glycosylation within the antibody population. A monoclonal antibody can be monospecific or polyspecific, or monovalent, bivalent or polyvalent. Bispecific antibodies are included within the term monoclonal antibody.

[0080] An "isolated antibody" refers to an antibody or antibody fragment that is substantially free of other antibodies having different antigen specificities. An "isolated antibody" encompasses antibodies that have been isolated to greater purity, such as antibodies that are at least 80%, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure.

[0081] "Humanized antibody" refers to an antibody in which the antigen binding site is derived from a non-human species and the variable region framework is derived from a human immunoglobulin sequence. Humanized antibodies can contain substitutions in the framework, and the framework need not be an exact copy of an expressed human immunoglobulin or human immunoglobulin germline gene sequence.

[0082] "Human antibody" refers to an antibody having heavy and light chain variable regions in which both the framework and antigen binding sites are derived from sequences of human origin, and is optimized to generate a minimal immune response when administered to a human subject. If the antibody contains a constant region or a portion of a constant region, the constant region also is derived from sequences of human origin.

[0083] "Anti-target" refers to an antibody or antibody domain (also called an antigen-binding portion or fragment of an antibody) that can bind to a specific target molecule such as CD38 (i.e., an anti-CD38 is an antibody or antibody domain that can bind to CD38). The format "CD38" refers to the CD38 protein or CD38 gene product. The format "CD38" refers to the gene for CD38.

[0084] "CD3xCD38" refers to a bispecific antibody or antibody fragment capable of binding to CD3 and CD38. The process of making a bispecific antibody requires recombinant modifications to either of the parent mAb amino acid sequences. Although the amino acid sequences of the CH1, CL, and Fc domains of each parent mAb must not be the same, there is no significant difference in binding between CD3xCD38 and CD38xCD3 bispecific antibodies. The notation of "first arm" and "second arm" herein is arbitrary. For example, in the bispecific antibodies disclosed herein, the first arm may target CD3 and the second arm may target CD38, or the first arm may target CD38 and the second arm may target CD3.

[0085] Numbering of amino acid residues in antibody constant regions throughout this specification is according to the EU index as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), unless expressly stated otherwise.

[0086] Conventional one-letter and three-letter amino acid codes, as shown in Table 1, are used herein. [Table 1]

[0087] The polypeptides, nucleic acids, fusion proteins and other compositions provided herein can include polypeptides, nucleic acids, fusion proteins, etc. that have a percentage identity recited to the amino acid or DNA sequences provided herein. The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent identity", "percent homology", "sequence identity" or "sequence homology", etc., refer to the percentage of identical residues between the amino acids or nucleotides in the compared molecules, which is calculated based on the size of the smallest molecule being compared. For example, sequence A that is "at least 85% identical" to sequence B means that sequence A contains at least 85%, e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, identical residues to the residues of sequence B. For such calculations, gaps in the alignment, if any, are preferably addressed by a particular mathematical model or computer program (ie, an "algorithm").Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073. In calculating percent identity, the sequences being compared are typically aligned in a way that gives the maximum match between the sequences.

[0088] The constant region sequence of a mammalian IgG heavy chain is H1 - Hinge-C H2 -C H3 The "hinge", "hinge region" or "hinge domain" of an IgG is generally defined as comprising Glu216 and terminating at Pro230 of human IgG1 according to the EU index, although functionally the flexible portion of the chain can be considered to include additional residues designated the upper and lower hinge regions, such as Glu216 to Gly237, with the lower hinge generally designated F. c F responsible for γR binding cThe hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form inter-heavy chain S-S bonds. The boundaries, numbered according to the EU index, may vary slightly, but are consistent with the C H1 The domain includes the VH domain and is adjacent to the amino terminus of the hinge region of an immunoglobulin heavy chain molecule and includes the first (most amino terminal) constant region of the immunoglobulin heavy chain, e.g., about EU positions 118-215. c The domain extends from amino acid 231 to amino acid 447; H2 The domain consists of approximately Ala231 to Lys340 or Gly341, C H3 extends from about Gly341 or Gln342 to Lys447. H1 The IgG heavy chain constant region residues of the region end with Lys. c The domain-containing molecule comprises at least C H2 Domain and C H3 domain and thus includes at least about the region from Ala231 to Lys447 of the IgG heavy chain constant region. c The domain-containing molecule can optionally include at least a portion of a hinge region.

[0089] "Epitope" refers to the portion of an antigen (e.g., CD3 or CD38) to which an antibody specifically binds. Epitopes typically consist of chemically active (such as polar, non-polar or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and can have specific charge characteristics as well as specific three-dimensional structural features. Epitopes can be composed of contiguous and / or discontinuous amino acids that form a conformational spatial unit. For discontinuous epitopes, amino acids from different parts of the linear sequence of the antigen are brought into close proximity in three-dimensional space due to folding of the protein molecule. Antibody "epitopes" depend on the methodology used to identify the epitope.

[0090] A "leader sequence" (also called a "signal peptide" or "signal sequence"), as used herein, includes any signal peptide that can be processed by a mammalian cell, including the human B2M leader. Such sequences are well known in the art.

[0091] A "cleavable linker" (also called a "protease sequence") is a peptide substrate that is cleavable by an enzyme. When operable, the cleavable linker is cleaved by an enzyme to allow activation of a shielded antibody (also called a pro-antibody) having a masking domain, e.g., an IGF2-based masking domain. Preferably, the cleavable linker is selected such that activation occurs at a desired site of action (in or near a target cell (e.g., a carcinoma cell) or tissue). For example, the cleavable linker is a peptide substrate specific for an enzyme that is specifically or highly expressed at the site of action, such that the cleavage rate of the cleavable linker at the target site is higher than at sites other than the target site.

[0092] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. These terms also include polypeptides having co-translational (e.g., signal peptide cleavage) and post-translational modifications of the polypeptide, such as, for example, disulfide bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage, and the like.

[0093] Furthermore, as used herein, "polypeptide" refers to a protein that contains modifications, such as deletions, additions and substitutions (generally conservative in nature, as known to those of skill in the art), relative to the native sequence, so long as the protein retains a desired activity. These modifications may be deliberate, such as by site-directed mutagenesis, or may be accidental, such as by mutations of hosts which produce the protein or by errors due to PCR amplification or other recombinant DNA methods.

[0094] The term "masking domain" (also called "shield", "shielding domain" or "mask") in this disclosure refers to a protein domain that can be fused to an antibody and that masks the antibody when it binds to its antigen. The shielding domain can mask the antibody from recognizing its target epitope, thus keeping the antibody in an inactive, shielded antibody form. Removal of the shielding domain exposes the variable domain of the antibody to bind to its target and exert its effect.

[0095] The term "recombinant," as used herein to describe a nucleic acid molecule, means a polynucleotide of genomic, cDNA, viral, semisynthetic and / or synthetic origin that is not related by its origin or manipulation to all or a portion of the polynucleotide sequence with which it is associated in nature. The term "recombinant," as used with respect to a protein or polypeptide, refers to a polypeptide produced by expression from a recombinant polynucleotide. The term "recombinant," as used with respect to a host cell or virus, refers to a host cell or virus into which a recombinant polynucleotide has been introduced. Recombinant is also used herein with respect to materials (e.g., cells, nucleic acids, proteins, or vectors) to refer to the material being altered by the introduction of heterologous material (e.g., cells, nucleic acids, proteins, or vectors).

[0096] The terms "polynucleotide," "oligonucleotide," "nucleic acid," and "nucleic acid molecule" are used interchangeably herein to include polymeric forms of nucleotides, either ribonucleotides or deoxyribonucleotides. The terms refer only to the primary structure of the molecule.

[0097] "Vector" refers to a polynucleotide that can be replicated within a biological system or can be transferred between such systems. A vector polynucleotide typically contains elements such as an origin of replication, a polyadenylation signal, or a selection marker that function to facilitate replication or maintenance of such polynucleotides in biological systems, such as cells, viruses, animals, plants, and reconstituted biological systems, utilizing biological components that can replicate vectors. A vector polynucleotide may be a single-stranded or double-stranded DNA or RNA molecule, cDNA, or a hybrid thereof.

[0098] An "expression vector" refers to a vector that can be utilized in a biological system or reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0099] As used herein, the term "heterologous" used in reference to a nucleic acid sequence, protein or polypeptide means that these molecules do not naturally occur in the cell from which the heterologous nucleic acid sequence, protein or polypeptide is obtained. For example, a nucleic acid sequence encoding a human polypeptide inserted into a cell that is not a human cell is a heterologous nucleic acid sequence in this particular context. While heterologous nucleic acids can be derived from different organisms or animal species, such nucleic acids do not necessarily originate from separate organism species to be heterologous. For example, in some instances, a synthetic nucleic acid sequence or its encoded polypeptide may be heterologous to the cell into which it is introduced in that the cell into which it is introduced did not previously contain the synthetic nucleic acid. Thus, for example, a synthetic nucleic acid sequence or its encoded polypeptide can be considered heterologous to a human cell even if one or more components of the synthetic nucleic acid sequence or its encoded polypeptide were originally obtained from a human cell.

[0100] "Host cell" as used herein means any type of cell that can be transformed with a nucleic acid or vector of the present disclosure to produce a polypeptide encoded thereby. For example, a host cell refers to a eukaryotic cell in vivo or in vitro, or a cell derived from a multicellular organism cultured as a single-cell entity (e.g., a cell line), and such eukaryotic cells can be or have been used as recipients of a nucleic acid (e.g., an expression vector comprising a nucleotide sequence encoding a multimeric polypeptide of the present disclosure), including the progeny of the original cell genetically modified by the nucleic acid. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or genomic or total DNA content to the original parent due to natural, accidental or deliberate mutation. A "recombinant host cell" (also referred to as a "genetically modified host cell") is a host cell into which a heterologous nucleic acid, e.g., an expression vector, has been introduced. For example, a genetically modified eukaryotic host cell has been genetically modified by the introduction into a suitable eukaryotic host cell of heterologous nucleic acid, e.g., exogenous nucleic acid that is foreign to the eukaryotic host cell, or recombinant nucleic acid that is not normally found in the eukaryotic host cell.

[0101] "Specific binding" or "specifically binds" or "binds" refers to an antibody binding to a specific antigen with greater affinity than to other antigens. Typically, the equilibrium dissociation constant (KD) for binding is about 1×10 -8 M or less, e.g., about 1×10 -9 M or less, approximately 1 x 10 -10 M or less, approximately 1 x 10 -11 M or less, or about 1×10 -12 An antibody "specifically binds" if it has a KD of M or less, typically at most one percent lower than its KD for binding to a nonspecific antigen (e.g., BSA, casein). KD can be measured using standard procedures.

[0102] As used herein, the terms "treatment", "treating" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or its symptoms, and / or therapeutic in terms of partially or completely curing the disease and / or adverse effects that may result from the disease. "Treatment", as used herein, encompasses any treatment of disease in a mammal, e.g., a human, and includes (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., arresting its development; and (c) alleviating the disease, i.e., causing regression of the disease.

[0103] The terms "individual," "subject," "host," and "patient," used interchangeably herein, refer to mammals, including but not limited to murines (e.g., rats, mice), lagomorphs (e.g., rabbits), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cows, sheep, pigs, goats), and the like.

[0104] A "therapeutically effective amount," "pharmaceutical effective amount," "effective amount," or "effective amount" refers to an amount of an agent, or a combined amount of two agents, that, when administered to a mammal or other subject for treating a disease, is sufficient to affect such treatment for the disease. A "therapeutically effective amount" will vary depending on the agent, the disease and its severity, and the age, weight, etc., of the subject to be treated.

[0105] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to the particular embodiments described, as they may, of course, vary. The scope of the present disclosure will be limited only by the appended claims, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. CD38 and anti-CD38 antibodies

[0106] As shown in FIG. 1A, CD38 is expressed primarily on immune cells and binds nicotinamide nucleotides (NADs). + and NMN) to ADPR and cADPR (Hogan, Chini et al. 2019). These metabolites mobilize calcium ions. Although intracellular CD38 is present in the cytoplasm and in the membranes of organelles, the majority of CD38 activity is extracellular and is due to the uptake of NAD + NAD synthesis required + As shown in Figure 1B, the extracellular activity of CD38 leads to the degradation of precursors (e.g., NMN) and NAD in the context of infection, metabolic dysfunction, aging, and tumor biology. +This has far-reaching implications for homeostasis. Multiple immune suppressive mechanisms thwart antitumor immunity. Among them, accumulation of extracellular adenosine is a potent and widespread strategy used by tumors to escape immune surveillance by activating purinergic receptors. In the immune system, attraction of A2a and A2b adenosine receptors is a critical regulatory mechanism that protects tissues against excessive immune responses. In tumors, this pathway is hijacked and antitumor immunity is impeded, promoting cancer progression (Allard, Beavis et al. 2016).

[0107] As shown in Figure 2, CD38 expression is higher in the thymus and prostate. However, expression levels are very high in activated BDCA4+ dendritic cells, CD56+ NK cells, leukemia cells, and lymphoma cells. This figure is generated using BioGPS software (Su, Wiltshire et al. 2004, Wu, Jin et al. 2016).

[0108] As shown in Figure 3, anti-CD38 antibodies can recognize CD38 on MM cells and provide anti-MM activity via Fc-dependent mechanisms and through immunomodulatory effects (Saltarella, Desantis et al. 2020). Fc-dependent mechanisms can include (a) antibody-dependent cellular cytotoxicity (ADCC); (b) antibody-dependent cellular phagocytosis (ADCP) via attraction of antibody Fc domains to effector cells expressing FcγR (e.g., NK cells, γδ T cells, neutrophils, and macrophages), which respectively cause lysis and / or phagocytosis of MM cells; (c) complement-dependent cytotoxicity (CDC) via attraction of C1q, which activates the complement cascade resulting in the assembly of membrane attack complexes (MACs) that can lyse target cells. Anti-CD38 antibodies can have immunomodulatory effects through inhibition of the extracellular enzymatic activity of CD38, resulting in a reduction of immunosuppressive extracellular adenosine (ADO). In addition, there is elimination of CD38+ immunosuppressive cells (i.e., MDSC, Treg, and Breg), which may result in enhanced T cell proliferation and effector function.

[0109] As shown in FIG. 4, anti-CD38 antibodies have the following effects: (a) clonal selection of CD38dim MM cells, thereby minimizing mAb mechanisms in cells; (b) reduction of CD38 through CD38 endocytosis, trogocytosis by granulocytes and monocytes, and through release of CD38-expressing microvesicles that contribute to adenosine production and immunosuppression; (c) immunomodulatory effects through downregulation of intracellular pathways in bone marrow-derived stem / stromal cells (BMSCs), reducing effector memory T cells, M1 macrophages, and costimulatory CD28 expression in T cells; (d) overexpression of CD46 and membrane-bound complement inhibitory proteins (CD55 and CD59) in MM cells, preventing CDC; (e) MM cell overexpression of CD47, which recognizes immune checkpoint signal regulatory protein α (SIRP) in tumors, thereby inhibiting antibody-dependent cellular phagocytosis (ADCP); and (f) inhibition of CD47 expression in tumors. Several resistance mechanisms may be induced, including loss of CD38+ NK cells by fratricidal ADCC.

[0110] The primary amino acid sequence of human CD38 is set forth in SEQ ID NO:1 of Table 2. Table 2 [Table 2]

[0111] In some embodiments, the disclosure provides anti-CD38 antibodies and antigen-binding portions thereof. As non-limiting examples, the disclosure provides anti-CD38 heavy and light chain variable region amino acid sequences set forth in Table 3 as SEQ ID NOs:2-30.

[0112] In some embodiments, the disclosure provides an anti-CD38 antibody, or antigen-binding portion thereof, comprising a heavy chain variable region comprising three complementarity determining regions (CDRs) denoted HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are selected from the following, respectively: SEQ ID NOs: 64, 65, and 66; SEQ ID NOs: 64, 83, and 84; SEQ ID NOs: 64, 65, and 85; and SEQ ID NOs: 64, 65, and 86.

[0113] In some embodiments, the disclosure provides an anti-CD38 antibody, or antigen-binding portion thereof, comprising a light chain variable region comprising three CDRs denoted LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 are selected from the following, respectively: SEQ ID NOs: 61, 62, and 63; SEQ ID NOs: 76, 77, and 78; SEQ ID NOs: 79, 80, and 81; and SEQ ID NOs: 82, 62, and 78.

[0114] In some embodiments, the disclosure provides an anti-CD38 antibody, or antigen-binding portion thereof, comprising a heavy chain variable region comprising three complementarity determining regions (CDRs) denoted HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are selected from the following, respectively: SEQ ID NOs: 64, 65, and 66; SEQ ID NOs: 64, 83, and 84; SEQ ID NOs: 64, 65, and 85; and SEQ ID NOs: 64, 65, and 86; and a light chain variable region comprising three CDRs denoted LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 are selected from the following, respectively: SEQ ID NOs: 61, 62, and 63; SEQ ID NOs: 76, 77, and 78; SEQ ID NOs: 79, 80, and 81; and SEQ ID NOs: 82, 62, and 78.

[0115] In some embodiments, the present disclosure provides an anti-CD38 antibody, or antigen-binding fragment thereof, comprising a heavy chain sequence comprising an amino acid sequence having at least 85% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of SEQ ID NOs: 12-16, or an antigen-binding portion thereof, and a light chain sequence comprising an amino acid sequence having at least 85% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of SEQ ID NOs: 7-11, or an antigen-binding portion thereof.

[0116] In some embodiments, the disclosure provides anti-CD38 antibodies, or antigen-binding fragments thereof, comprising light chain and heavy chain sequences comprising the following, respectively: SEQ ID NOs:2 and 3; SEQ ID NOs:7 and 12; SEQ ID NOs:7 and 13; SEQ ID NOs:7 and 14; SEQ ID NOs:7 and 15; SEQ ID NOs:7 and 16; SEQ ID NOs:8 and 12; SEQ ID NOs:8 and 13; SEQ ID NOs:8 and 14; SEQ ID NOs:8 and 15; SEQ ID NOs:8 and 16; SEQ ID NOs:9 and 12; SEQ ID NOs:9 and 13; SEQ ID NOs:9 and 14; SEQ ID NOs:9 and 15; SEQ ID NOs:9 and 16; SEQ ID NOs:10 and 12; SEQ ID NOs:10 and 13; SEQ ID NOs:10 and 14; SEQ ID NOs:10 and 15; SEQ ID NOs:10 and 16; SEQ ID NOs:11 and 12; SEQ ID NOs:11 and 13; SEQ ID NOs:11 and 14; SEQ ID NOs:11 and 15; or SEQ ID NOs:11 and 16.

[0117] In some embodiments, the disclosure comprises at least one variable heavy chain-only single domain or antigen-binding portion thereof, wherein the at least one variable heavy chain-only single domain is selected from the following, respectively: SEQ ID NOs: 67, 68, and 69; SEQ ID NOs: 67, 87, and 69; SEQ ID NOs: 67, 88, and 69; SEQ ID NOs: 67, 89, and 69; SEQ ID NOs: 67, 68, and 90; SEQ ID NOs: 70, 91, and 72; SEQ ID NOs: 70, 92, and and 72; SEQ ID NOs: 70, 93, and 72; SEQ ID NOs: 94, 95, and 96; SEQ ID NOs: 70, 71, and 97; SEQ ID NOs: 73, 98, and 75; SEQ ID NOs: 73, 99, and 75; SEQ ID NOs: 73, 100, and 75; SEQ ID NOs: 73, 101, and 102; and SEQ ID NOs: 73, 103, and 104.

[0118] In some embodiments, the disclosure provides an anti-CD38 antibody, or antigen-binding portion thereof, comprising at least one variable heavy-only single domain, or antigen-binding portion thereof, wherein the at least one variable heavy-only (VHO) single domain comprises an amino acid sequence, or antigen-binding portion thereof, having at least 85% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of SEQ ID NOs: 4, and 17-30.

[0119] The anti-CD38 antibodies of the present disclosure include full-length antibodies comprising two heavy chains and two light chains. The antibody may be a human or humanized chimeric antibody. Humanized antibodies, as used herein, include chimeric and CDR-grafted antibodies. Chimeric antibodies are antibodies comprising a non-human antibody variable region linked to a human constant region. CDR-grafted antibodies are antibodies comprising CDRs from a non-human "donor" antibody linked to framework regions from a human "recipient" antibody. Exemplary human or humanized antibodies include IgG, IgM, IgE, IgA and IgD antibodies. The antibody may be of any class (e.g., IgG, IgM, IgE, IgGA, IgD) or isotype. For example, a human antibody may comprise an IgG Fc domain, such as at least one of the isotypes IgG1, IgG2, IgG3 or IgG4.

[0120] In some embodiments, the anti-CD38 antibody or antigen-binding portion thereof is selected from the group consisting of a whole antibody, an antibody fragment, a human antibody, a humanized antibody, a chimeric antibody, a single chain antibody, a conjugate, an antibody mimic, and a defucosylated antibody. In further examples, the anti-CD38 antibody fragment is selected from the group consisting of a UniBody, a single domain antibody with only a variable heavy chain, and a Nanobody. For example, the anti-CD38 antibody fragment is a Nanobody set forth in SEQ ID NOs: 17-30. In some examples, the anti-CD38 antibody fragment is selected from the group consisting of a single domain VHH, a single domain VHO, an Affibody, a DARPin, an Anticalin, an Avimer, a Versa body, and a Duocalin. Anti-CD3 antibody

[0121] In some embodiments, the disclosure provides anti-CD3 antibodies and antigen-binding portions thereof. As non-limiting examples, the disclosure provides the anti-CD3 amino acid sequences set forth in Table 4 as SEQ ID NOs: 31-41.

[0122] In some embodiments, the disclosure provides an anti-CD3 antibody, or antigen-binding portion thereof, comprising a heavy chain variable region comprising three complementarity determining regions (CDRs) denoted HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are selected from the following, respectively: SEQ ID NOs: 105, 108, and 107; SEQ ID NOs: 105, 109, and 107; SEQ ID NOs: 105, 110, and 107; and SEQ ID NOs: 118, 119, and 120.

[0123] In some embodiments, the disclosure provides an anti-CD3 antibody, or an antigen-binding portion thereof, comprising a light chain variable region comprising three CDRs designated LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 are selected from the following: SEQ ID NOs: 114, 112, and 113; and SEQ ID NOs: 115, 116, and 117, respectively.

[0124] In some embodiments, the disclosure provides an anti-CD3 antibody, or antigen-binding portion thereof, comprising a heavy chain variable region comprising three complementarity determining regions (CDRs) denoted HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are selected from the following, respectively: SEQ ID NOs: 105, 108, and 107; SEQ ID NOs: 105, 109, and 107; SEQ ID NOs: 105, 110, and 107; and SEQ ID NOs: 118, 119, and 120; and a light chain variable region comprising three CDRs denoted LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 are selected from the following, respectively: SEQ ID NOs: 114, 112, and 113; and SEQ ID NOs: 115, 116, and 117.

[0125] In some embodiments, the present disclosure provides an anti-CD3 antibody, or antigen-binding fragment thereof, comprising a heavy chain sequence comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 32-34, and 39, or an antigen-binding portion thereof, and a light chain sequence comprising an amino acid sequence having at least 85% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of SEQ ID NOs: 36-38, or an antigen-binding portion thereof.

[0126] In some embodiments, the disclosure provides anti-CD3 antibodies, or antigen-binding fragments thereof, comprising light chain and heavy chain sequences comprising the following, respectively: SEQ ID NOs: 39 and 38; SEQ ID NOs: 41 and 40; SEQ ID NOs: 31 and 35; SEQ ID NOs: 31 and 36; SEQ ID NOs: 31 and 37; SEQ ID NOs: 32 and 35; SEQ ID NOs: 32 and 36; SEQ ID NOs: 32 and 37; SEQ ID NOs: 33 and 35; SEQ ID NOs: 33 and 36; SEQ ID NOs: 33 and 37; SEQ ID NOs: 34 and 35; SEQ ID NOs: 34 and 36; or SEQ ID NOs: 34 and 37.

[0127] The anti-CD3 antibodies of the present disclosure include full-length antibodies comprising two heavy chains and two light chains. The antibodies may be human or humanized.

[0128] In some embodiments, the anti-CD3 antibody or antigen-binding portion thereof is selected from the group consisting of a whole antibody, an antibody fragment, a human antibody, a humanized antibody, a single chain antibody, a conjugate, an antibody mimetic, and a defucosylated antibody. In further examples, the anti-CD3 antibody fragment is selected from the group consisting of a UniBody, a single domain antibody with only a variable heavy chain, and a Nanobody. In some examples, the anti-CD3 antibody fragment is selected from the group consisting of a single domain VHH, a single domain VHO, an Affibody, a DARPin, an Anticalin, an Avimer, a Versa body, and a Duocalin. Bispecific CD3×CD38 antibody

[0129] In some embodiments, the disclosure provides CD3×CD38 bispecific antibodies that simultaneously target proteins linked to CD38-related pathways as well as proteins capable of activating CD3 T cell activity. In some embodiments, the disclosure provides CD3×CD38 bispecific antibodies that include a shielding domain. In some embodiments, the disclosure provides CD3×CD38 bispecific antibodies without a shielding domain. CD3 and CD38 targets have differential expression levels at pathological sites and normal tissues. The shielded CD3×CD38 bispecific antibodies remain negatively active in normal tissues due to the inhibitory effect of the masking domain on the binding domain. The masking domain is cleaved off by proteases at the disease site, and the shielded CD3×CD38 bispecific antibody is converted to an active CD3×CD38 bispecific antibody.

[0130] In some embodiments, the present disclosure provides bispecific antibodies that simultaneously target and bind human CD38 and CD3, have high affinity, and can effectively block CD38 protein at the protein level. Bispecific antibodies have the ability to bind both CD3 and CD38 proteins and bind one protein without affecting the binding of the other protein, i.e., bind CD3 and CD38 simultaneously. The bispecific antibodies disclosed herein fill the gap where there are no antibodies that simultaneously target CD3 and CD38. The bispecific antibodies disclosed herein inhibit CD38-mediated diseases or disorders, such as proliferation of vascular endothelial cells, human lung cancer cells, human breast cancer cells, human pancreatic cancer cells, and / or human gastric cancer cells.

[0131] In some embodiments, the present disclosure provides the following: A first binding arm comprising: a first heavy chain fusion protein comprising, from N-terminus to C-terminus, shield A, protease sequence A, and an IgG heavy chain or an antigen-binding portion thereof; and a first light chain fusion protein comprising, from N-terminus to C-terminus, shield B, protease sequence B, and an IgG light chain or an antigen binding portion thereof; Contains; wherein the IgG heavy chain or antigen-binding portion thereof and the IgG light chain or antigen-binding portion thereof of the first binding arm are capable of targeting the CD3-related pathway; and A second binding arm comprising: a second heavy chain fusion protein comprising, from N-terminus to C-terminus, a shield C, a protease sequence C, and an IgG heavy chain or an antigen-binding portion thereof; and a second light chain fusion protein comprising, from N-terminus to C-terminus, a shield D, a protease sequence D, and an IgG light chain or an antigen-binding portion thereof; Contains; wherein the IgG heavy chain or an antigen-binding portion thereof and the IgG light chain or an antigen-binding portion thereof of the second binding arm are capable of targeting the CD38-related pathway. The present invention provides a bispecific antibody comprising: The shields A through D can be the same or different from each other, and the protease sequences A through D can be the same or different from each other.

[0132] In some embodiments, the present disclosure provides the following: A first binding arm comprising: a first heavy chain fusion protein comprising, from N-terminus to C-terminus, signal sequence A-shield A-linker A-protease sequence A-linker B-IgG heavy chain or antigen binding portion thereof; and A first light chain fusion protein comprising from N-terminus to C-terminus: signal sequence B-shield B-linker C-protease sequence B-linker D-IgG light chain or an antigen binding portion thereof; Contains; wherein the IgG heavy chain or antigen-binding portion thereof and the IgG light chain or antigen-binding portion thereof of the first binding arm are capable of targeting the CD3-related pathway; and A second binding arm comprising: A second heavy chain fusion protein comprising, from N-terminus to C-terminus, a signal sequence C-shield C-linker E-protease sequence C-linker F-IgG heavy chain or an antigen-binding portion thereof; and A second light chain fusion protein comprising from N-terminus to C-terminus: signal sequence, D-shield, D-linker, G-protease sequence, D-linker, H-IgG light chain, or an antigen-binding portion thereof; Contains; wherein the IgG heavy chain or an antigen-binding portion thereof and the IgG light chain or an antigen-binding portion thereof of the second binding arm are capable of targeting the CD38-related pathway. The present invention provides a bispecific antibody comprising: The signal sequences A through D can be the same or different from each other, and the linkers A through H can be the same or different from each other.

[0133] In some embodiments, the present disclosure provides the following: A first binding arm comprising: a first heavy chain fusion protein comprising, from N-terminus to C-terminus, shield A, protease sequence A, and an IgG heavy chain or an antigen-binding portion thereof; and a first light chain fusion protein comprising, from N-terminus to C-terminus, shield B, protease sequence B, and an IgG light chain or an antigen binding portion thereof; Contains; wherein the IgG heavy chain or antigen-binding portion thereof and the IgG light chain or antigen-binding portion thereof of the first binding arm are capable of targeting the CD3-related pathway; and A second binding arm comprising: a second heavy chain fusion protein comprising, from N-terminus to C-terminus, a shield C, a protease sequence C, and an IgG heavy chain (comprising at least one variable heavy chain only (VHO) single domain or antigen binding portion thereof); Contains; wherein a single domain of only said at least one variable heavy chain or an antigen-binding portion thereof is capable of targeting the CD38-related pathway; The present invention provides a bispecific antibody comprising: The shields A through C can be the same or different from each other, and the protease sequences A through C can be the same or different from each other.

[0134] In some embodiments, the present disclosure provides the following: A first binding arm comprising: a first heavy chain fusion protein comprising, from N-terminus to C-terminus, signal sequence A-shield A-linker A-protease sequence A-linker B-IgG heavy chain or antigen binding portion thereof; and A first light chain fusion protein comprising from N-terminus to C-terminus: signal sequence B-shield B-linker C-protease sequence B-linker D-IgG light chain or an antigen binding portion thereof; Contains; wherein the IgG heavy chain or antigen-binding portion thereof and the IgG light chain or antigen-binding portion thereof of the first binding arm are capable of targeting the CD3-related pathway; and A second binding arm comprising: A second heavy chain fusion protein comprising, from N-terminus to C-terminus, a signal sequence C-shield C-linker E-protease sequence C-linker F-IgG heavy chain (comprising at least one single domain of only the variable heavy chain or an antigen binding portion thereof); Contains; wherein at least one single domain of only the variable heavy chain is capable of targeting the CD38-related pathway; The present invention provides a bispecific antibody comprising: The signal sequences A to C can be the same or different from each other, and the linkers A to F can be the same or different from each other.

[0135] In some embodiments, a bispecific antibody consists of two sets of light chain fusions and two sets of heavy chain fusions. For example, the structures of light chain fusions and heavy chain fusions from each parent antibody are shown in FIG. 5. For example, a bispecific antibody may comprise a human IgG1 heavy chain fusion chain comprising the amino acid sequence of signal sequence A-shield A-linker A-protease sequence A-linker B-IgG1 heavy chain from N-terminus to C-terminus; and a human IgG1 light chain fusion comprising the amino acid sequence of signal sequence B-shield B-linker B-protease B-linker C-IgG1 light chain from N-terminus to C-terminus. Signal sequence A can be the same as or different from signal sequence B. Shield A can be the same as or different from shield B. Linker A can be the same as or different from linker B. Protease sequence B can be the same as or different from protease sequence A. In some embodiments, a bispecific antibody consists of two sets of heavy chain fusions and one set of light chain fusions as illustrated in Figures 6A and 6B.

[0136] The present disclosure provides bispecific antibodies that can be created using well-established point mutations in the CH1, CH2, and CH3 domains by controlled Fab arm exchange or by co-expression. In some embodiments, all constructs are symmetrical such that there is no preference regarding the choice of point mutations of each parent antibody.

[0137] Figures 5-7 illustrate several formats of bispecific antibodies disclosed herein with or without a masking domain. Figure 5A shows a bispecific antibody with two different sets of heavy chain (HC) and light chain (LC) combinations, annotated as "first arm" (targeting CD3) and "second arm" (targeting CD38). Figure 5B shows that the CD3 arm (first arm) of the bispecific Ab can be a human IgG with HC and LC, where the light chain fusion comprises, from N-terminus to C-terminus: mask domain B, protease cleavable linker B, LC variable region VL, and constant light chain CL (e.g., Cλ), and where the heavy chain fusion comprises, from N-terminus to C-terminus: mask domain A, protease cleavable linker A, HC comprises variable region VH, CH1 domain, and Fc region. Figure 5C shows that the CD38 binding arm (second arm) of the bispecific Ab can be a human IgG with HC and LC, where the light chain fusion comprises, from N-terminus to C-terminus: mask domain B, protease cleavable linker B, LC variable region VL, and constant light chain CL (e.g., Cκ or Cλ), and where the heavy chain comprises, from N-terminus to C-terminus: mask domain A, protease cleavable linker A, tandem HC variable region VH or VHO, CH1 domain, and Fc region. The Fc region can be further genetically fused to a homing domain (HD). Different or the same linkers can be placed between mask A and protease cleavable linker A, protease cleavable linker A and VH, and between the Fc domain and the HD domain. Different or the same linkers are placed between the mask B and the protease cleavable linker B, between the protease cleavable linker B and the VH or VHO in tandem, and between the Fc domain and the HD domain. The heavy and light chains may have the same or different complementary mask domains and protease cleavable linkers.

[0138] FIG. 6A shows that the bispecific antibody contains a single VHO in the "second arm", the arm that targets CD38, and FIG. 6B shows that the CD38 binding arm (second arm) contains two VHOs fused together. FIG. 6C shows that the CD3 arm (first arm) of the bispecific Ab illustrated in FIG. 6A and 6B can contain a human IgG with the HC and LC illustrated in FIG. 5B. FIG. 6C also shows that the CD38 binding arm contains, from the end to the C-terminus: mask domain A, protease cleavable linker A, HC VHO variable region, CH1 domain, and Fc region. Two or more VHO fusions separated by linker domains can exist for higher specificity. The CD38 binding arm can have a different linker and masking domain than the CD3 binding arm.

[0139] Figure 7 is a schematic diagram showing removal of the masking domain from a CD38xCD3 bispecific antibody. Proteases present at high concentrations in the tumor microenvironment can cleave along the protease-cleavable linker to convert the shielded bispecific antibody into an active bispecific antibody.

[0140] For example, genetic fusion of the VHO domain of the bispecific antibodies disclosed herein, comprising a human VHO single domain linked to either the CH1-hinge-CH2-CH3 domain of human IgG1 or the hinge-CH2-CH3 domain of human IgG1, results in a highly soluble chimeric heavy chain antibody with half the size of conventional antibodies (75 vs. 150 kDa), as shown in Figures 5 and 6. Such constructs may penetrate tissues better than conventional antibodies. In addition, the bispecific molecules disclosed herein will take advantage of their additional tumor cell growth inhibitory efficacy in diseased tissues, further mitigating high toxicity concerns by shielding the binding epitopes of CD3 and CD38 in normal tissues, as shown in Figure 7. Thus, the bispecific antibodies disclosed herein provide effective targeting of CD38 for oncology and other disorders rooted in CD38 overexpression, albeit with reduced safety concerns, expanding the role of CD38 in the context of therapy for many cancers, as well as other non-oncology indications.

[0141] In some embodiments of the bispecific antibodies disclosed herein, the binding arm targeting CD3 has a valency of one. It is preferred that the bispecific antibodies are designed to have monovalent CD3 binding (i.e., one Fab arm that binds to an epitope on CD3). Bivalent CD3 binding is associated with excessive activation-induced cell death in effector cells that would limit the effectiveness of T cell redirection. In addition, bivalent CD3 redirecting molecules result in tumor antigen-independent immune effector cell activation that may enhance systemic toxicity in patients. The use of high affinity anti-CD3 antibody Fab arms may also enhance toxicity. High affinity variants of anti-CD3 Fab or scFv are usually difficult to tolerate in cynomolgus monkeys due to the resulting large cytokine release. High affinity to CD3 also shifted the biodistribution of the bispecific antibody from tumors to CD3-rich tissues, leading to a high risk of cytokine release syndrome. Thus, it is highly desirable that the binding affinity of the anti-CD3 arm is lower than that of the anti-CD38 arm. Bivalent anti-CD3 agents can have increased affinity in binding to CD3 and may also lead to increased cytokine release syndrome from non-tumor CD3 positive cells.

[0142] In some embodiments of the bispecific antibodies disclosed herein, the second binding arm targeting a CD38-related pathway, such as CD38, may comprise two to four, such as two or three, tandem, IgG variable heavy chain-only single domains or antigen-binding portions thereof, where the two or three IgG variable heavy chain-only single domains or antigen-binding portions thereof are optionally connected via one or more linkers. The second binding arm targeting a CD38-related pathway, such as CD38, may be monovalent, bivalent, trivalent, tetravalent, etc. For example, there may be dual, triple, or quadruple epitope recognition for CD38 in the bispecific antibody to increase the specificity of attraction.

[0143] Long-term administration of anti-CD3 or anti-CD38 biologic drugs poses a significant risk factor for patients. In some embodiments, the present disclosure provides a combination of shields that can form intermolecular interactions to prevent Fab arm attraction to their respective epitopes. The shields can be fused to the heavy and / or light chain domains. The shields can prevent the CDR regions from binding to antigens by Fab steric hindrance. The presence of the shields can minimize the systemic toxicity of the CD3×CD38 bispecific antibodies disclosed herein and can enhance the safety profile and therapeutic window of each arm of the bispecific antibody. The shields can be removed by proteases found in the tumor microenvironment and / or other enzymes specific to the site. The shielding domains (also called shields, masks, or masking domains) of the bispecific antibodies disclosed herein, e.g., shields A, B, C, D, E, and F, can be the same or different. In some embodiments, shield A, shield B, shield C, shield D, shield E, and shield F are each independently selected from the amino acid sequences set forth in SEQ ID NOs: 42-52.

[0144] The protease sequences of the bispecific antibodies disclosed herein can be the same or different, e.g., protease sequences A, B, C, D, E, and F. In some embodiments, protease sequence A, protease sequence B, protease sequence C, protease sequence D, protease sequence E, and protease sequence F are each independently selected from the amino acid sequences set forth in SEQ ID NOs: 53-60.

[0145] The peptide linkers of the bispecific antibodies disclosed herein, e.g., linkers A, B, C, D, E, and F, can be the same or different.

[0146] In some embodiments, the bispecific antibody disclosed herein comprises a heavy chain sequence selected from SEQ ID NOs: 3 and 12-16, and a light chain sequence selected from SEQ ID NOs: 2 and 7-11.

[0147] In some embodiments, the bispecific antibodies disclosed herein may comprise multiple binding arms targeting CD38. For example, the bispecific antibodies disclosed herein comprise a first CD38 binding arm comprising a light chain comprising SEQ ID NO:2 and a heavy chain comprising SEQ ID NO:3; a second CD38 binding arm comprising a light chain sequence selected from SEQ ID NOs:7-11 and a heavy chain sequence selected from SEQ ID NOs:12-16.

[0148] In some embodiments, a bispecific antibody disclosed herein comprises a binding arm capable of targeting CD38, wherein the binding arm comprises human IgG1 heavy and light chain sequences selected from SEQ ID NOs: 2-6. Leader sequence

[0149] In certain embodiments, a leader peptide is incorporated to drive secretion of the antibodies described herein, e.g., shielded CD3xCD38 bispecific antibodies, as the respective parent antibody proteins are secreted into the cell culture supernatant. Any leader peptide for any known secreted protein / peptide can be used.

[0150] As used herein, a "leader peptide," "lead peptide," or "signal peptide" refers to a short peptide, typically 16-30 amino acids in length, that is present at the N-terminus of most newly synthesized proteins destined for the secretory pathway. Although lead peptides are extremely heterogeneous in sequence, and many prokaryotic and eukaryotic lead peptides are functionally interchangeable even between different species, the efficiency of protein secretion can be strongly determined by the sequence of the lead / signal peptide.

[0151] In certain embodiments, the leader peptide is derived from a protein that resides inside either a particular cellular organelle (such as the endoplasmic reticulum, the Golgi apparatus, or an endosome), is secreted from the cell, or is inserted into most cell membranes.

[0152] In certain embodiments, the leader peptide is derived from a eukaryotic protein.

[0153] In certain embodiments, the leader peptide is derived from a secreted protein, eg, a protein that is secreted outside of a cell.

[0154] In certain embodiments, the leader peptide is derived from a transmembrane protein.

[0155] In certain embodiments, the leader peptide comprises a stretch of amino acids that is recognized and cleaved by a signal peptidase.

[0156] In certain embodiments, the leader peptide does not include a signal peptidase cleavage recognition sequence.

[0157] In certain embodiments, the leader peptide is a signal peptide for tissue plasminogen activator (tPA), herpes simplex virus glycoprotein D (HSV gD), growth hormone, a cytokine, lipoprotein transport signal, CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD4, CD8 alpha, CD19, CD28, 4-1BB, or GM-CSFR, or the S. cerevisiae mating factor alpha-1 signal peptide.

[0158] In some embodiments, the leader sequence described herein may be a mammalian CD4 or CD8 leader sequence, including, but not limited to, for example, a human CD4 or CD8 leader sequence, a non-human primate CD4 or CD8 leader sequence, a rodent CD4 or CD8 leader sequence, etc. In some embodiments, the CD4 or CD8 leader comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a human CD4 or CD8 leader sequence. Anti-CD3 and anti-CD38 antibodies and fragments applicable to shielded CD3xCD38 bispecific antibody design

[0159] In some embodiments, the present disclosure provides anti-CD3 and anti-CD38 antibodies and antigen-binding fragments thereof for CD3xCD38 bispecific antibody design. The fabs of CD3 and CD38 antibodies or antigen-binding fragments thereof can be attached to a shield via a protease cleavable linker sequence to generate the shielded CD3xCD38 bispecific antibody disclosed herein.

[0160] Anti-CD3 and anti-CD38 antibodies and fragments applicable to the shielded CD3xCD38 bispecific antibody design of the present disclosure include full-length antibodies comprising two heavy chains and two light chains. The antibodies can be human or humanized.

[0161] In some embodiments, the anti-CD38 or anti-CD3 antibody is selected from the group consisting of a whole antibody, an antibody fragment, a human or humanized antibody, a single chain antibody, a conjugate, an antibody mimic, and a defucosylated antibody. In further examples, the anti-CD38 or anti-CD3 antibody fragment is selected from the group consisting of a UniBody, a single domain antibody with only a variable heavy chain, and a Nanobody. For example, the anti-CD38 antibody fragment is a Nanobody set forth in SEQ ID NOs: 17-30. In some examples, the anti-CD38 or anti-CD3 antibody is selected from the group consisting of a single domain VHH, a single domain VHO, an Affibody, a DARPin, an Anticalin, an Avimer, a Versa body, and a Duocalin. As a non-limiting example, the present disclosure provides anti-CD38 heavy and light chain variable region amino acid sequences set forth in Table 3 as SEQ ID NOs: 2-30. As a non-limiting example, the present disclosure provides CD3 construct amino acid sequences set forth in Table 4 as SEQ ID NOs: 31-41. The bispecific antibodies disclosed herein can be made comprising any suitable combination of the amino acid sequences set forth in Tables 3 and 4. Table 3 [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] Table 4 [Table 4-1] [Table 4-2] [Table 4-3]

[0162] In some embodiments, the disclosure provides an isolated anti-CD38 monoclonal antibody, or an antigen-binding portion thereof, antibody fragment, or antibody mimetic, that binds to an epitope on human CD38 recognized by an antibody comprising a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 3, and 12-16, and a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 2, and 7-11.

[0163] In some embodiments, the disclosure provides an anti-CD38 antibody selected from the group consisting of a single domain VHH, a single domain VHO (variable heavy only) domain comprising a HC (heavy chain) variable region comprising an amino acid sequence selected from SEQ ID NOs: 4-6 and 17-30, an Affibody, a DARPin, an Anticalin, an Avimer, a Versabody, and a Duocalin.

[0164] In some embodiments, the disclosure provides a composition comprising an isolated anti-CD38 antibody, or antigen-binding portion, antibody fragment, or antibody mimetic disclosed herein, and a pharma- ceutically acceptable carrier.

[0165] In some embodiments, the disclosure provides an isolated nucleic acid molecule encoding a heavy or light chain or an antigen-binding portion of an isolated anti-CD38 antibody, or an antibody fragment that binds an epitope on human CD38. In some embodiments, the disclosure provides an expression vector comprising such a nucleic acid molecule, and a host cell comprising such an expression vector.

[0166] In some embodiments, the disclosure provides methods of preparing an anti-CD38 antibody, the method comprising the steps of obtaining host cells comprising one or more nucleic acid molecules encoding an anti-CD38 antibody; culturing the host cells in a host cell culture; providing host cell culture conditions wherein the one or more nucleic acid molecules are expressed; and recovering the antibody from the host cells or from the host cell culture.

[0167] Also described is an isolated anti-CD38 monoclonal antibody, or antigen-binding portion thereof, antibody fragment, or antibody mimetic, that binds an epitope on human CD38 that is recognized by an antibody comprising a heavy chain variable region and a light chain variable region selected from the group consisting of: a heavy chain variable region amino acid sequence set forth in SEQ ID NOs: 3, and 12-16, and a light chain variable region amino acid sequence set forth in SEQ ID NOs: 2, and 7-11; and a heavy chain single domain variable region amino acid sequence set forth in SEQ ID NOs: 4-6, and 17-30.

[0168] Also described herein is an isolated monoclonal antibody, or antigen-binding portion thereof, that binds an epitope on a CD38 polypeptide having the amino acid sequence of SEQ ID NO: 1 that is recognized by an antibody comprising a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 3 and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 2. Also described herein is an isolated monoclonal single domain antibody, or antigen-binding portion thereof, that binds an epitope on a CD38 polypeptide having the amino acid sequence of SEQ ID NO: 1 that is recognized by an antibody comprising a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 4-6 and 17-30.

[0169] The following: a set of heavy chain variable region amino acid sequences as defined in SEQ ID NO: 12 and light chain variable region amino acid sequences as defined in SEQ ID NO: 7; a set of heavy chain variable region amino acid sequences as defined in SEQ ID NO: 12 and light chain variable region amino acid sequences as defined in SEQ ID NO: 8; a set of heavy chain variable region amino acid sequences as defined in SEQ ID NO: 12 and light chain variable region amino acid sequences as defined in SEQ ID NO: 9; a set of heavy chain variable region amino acid sequences as defined in SEQ ID NO: 12 and light chain variable region amino acid sequences as defined in SEQ ID NO: 10; a set of heavy chain variable region amino acid sequences as defined in SEQ ID NO: 12 and light chain variable region amino acid sequences as defined in SEQ ID NO: 11; a set of heavy chain variable region amino acid sequences as defined in SEQ ID NO: 13 and light chain variable region amino acid sequences as defined in SEQ ID NO: 7; a set of heavy chain variable region amino acid sequences as defined in SEQ ID NO: 13 and light chain variable region amino acid sequences as defined in SEQ ID NO: 8; a set of heavy chain variable region amino acid sequences as defined in SEQ ID NO: 13 and light chain variable region amino acid sequences as defined in SEQ ID NO: 9; a set of heavy chain variable region amino acid sequences as defined in SEQ ID NO: 13 and light chain variable region amino acid sequences as defined in SEQ ID NO: 10 a set of a heavy chain variable region amino acid sequence defined in SEQ ID NO: 13 and a light chain variable region amino acid sequence defined in SEQ ID NO: 11; a set of a heavy chain variable region amino acid sequence defined in SEQ ID NO: 14 and a light chain variable region amino acid sequence defined in SEQ ID NO: 7; a set of a heavy chain variable region amino acid sequence defined in SEQ ID NO: 14 and a light chain variable region amino acid sequence defined in SEQ ID NO: 8; a set of a heavy chain variable region amino acid sequence defined in SEQ ID NO: 14 and a light chain variable region amino acid sequence defined in SEQ ID NO: 9; a set of a heavy chain variable region amino acid sequence defined in SEQ ID NO: 14 and a light chain variable region amino acid sequence defined in SEQ ID NO: 10; a set of a heavy chain variable region amino acid sequence defined in SEQ ID NO: 14 and a light chain variable region amino acid sequence defined in SEQ ID NO: 11; a set of a heavy chain variable region amino acid sequence defined in SEQ ID NO: 15 and a light chain variable region amino acid sequence defined in SEQ ID NO: 7; a set of a heavy chain variable region amino acid sequence defined in SEQ ID NO: 15 and a light chain variable region amino acid sequence defined in SEQ ID NO: 8; a set of a heavy chain variable region amino acid sequence defined in SEQ ID NO: 15 and a light chain variable region amino acid sequence defined in SEQ ID NO: 9;Also described is an isolated monoclonal antibody or antigen-binding portion thereof, antibody fragment, or antibody mimetic that binds an epitope on human CD38 recognized by an antibody comprising a heavy chain variable region and a light chain variable region selected from the group consisting of: a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region amino acid sequence set forth in SEQ ID NO: 10; a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region amino acid sequence set forth in SEQ ID NO: 11; a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 16 and a light chain variable region amino acid sequence set forth in SEQ ID NO: 7; a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 16 and a light chain variable region amino acid sequence set forth in SEQ ID NO: 8; a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 16 and a light chain variable region amino acid sequence set forth in SEQ ID NO: 9; a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 16 and a light chain variable region amino acid sequence set forth in SEQ ID NO: 10; and a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 16 and a light chain variable region amino acid sequence set forth in SEQ ID NO: 11.

[0170] Also described is an isolated monoclonal antibody or antigen-binding portion thereof, antibody fragment, or antibody mimetic that binds an epitope on human CD3 recognized by an antibody comprising a heavy chain variable region and a light chain variable region selected from the group consisting of the heavy chain and light chain variable region amino acid sequences set forth in the following: SEQ ID NO:31 and SEQ ID NO:35; SEQ ID NO:31 and SEQ ID NO:36; SEQ ID NO:31 and SEQ ID NO:37; SEQ ID NO:32 and SEQ ID NO:35; SEQ ID NO:32 and SEQ ID NO:36; SEQ ID NO:32 and SEQ ID NO:37; SEQ ID NO:33 and SEQ ID NO:35; SEQ ID NO:33 and SEQ ID NO:36; SEQ ID NO:33 and SEQ ID NO:37; SEQ ID NO:34 and SEQ ID NO:35; SEQ ID NO:34 and SEQ ID NO:36; SEQ ID NO:34 and SEQ ID NO:37; SEQ ID NO:39 and SEQ ID NO:38; SEQ ID NO:41 and SEQ ID NO:40.

[0171] In some embodiments, the present disclosure provides an isolated nucleic acid molecule encoding an isolated anti-CD38 heavy or light chain or anti-CD3 antibody of the present disclosure, or an antigen-binding portion thereof, and may in further aspects include an expression vector comprising such a nucleic acid and a host cell comprising such an expression vector.

[0172] Another embodiment of the present disclosure is a hybridoma expressing the disclosed anti-CD38 or anti-CD3 antibodies, or antigen-binding portions thereof.

[0173] Another aspect of the present disclosure is a method of making an anti-CD38 or anti-CD3 antibody of the present disclosure, comprising: immunizing an animal with a CD38 peptide or CD3 peptide; a CD38 protein or CD3 protein; or a CD38 domain or CD3 domain; recovering mRNA from B cells of said animal; and converting said mRNA into cDNA.

[0174] In some embodiments, the disclosure provides methods for preparing an anti-CD38 antibody or an anti-CD3 antibody, the method comprising the steps of obtaining a host cell comprising one or more nucleic acid molecules encoding an anti-CD38 antibody or an anti-CD3 antibody of the disclosure; culturing the host cell in a host cell culture; providing host cell culture conditions wherein the one or more nucleic acid molecules are expressed; and recovering the antibody from the host cell or from the host cell culture.

[0175] In some embodiments, the disclosure provides methods of expressing cDNAs encoding anti-CD38 or anti-CD3 monoclonal antibodies, or antigen-binding portions thereof, antibody fragments, or antibody mimetics, comprising expressing the cDNAs in phage (such that an anti-CD38 antibody or an anti-CD3 antibody encoded by the cDNA is displayed on the surface of the phage); selecting phage displaying anti-CD38 or anti-CD3 antibodies; recovering nucleic acid molecules from the selected phage encoding the anti-CD38 immunoglobulin and the anti-CD3 immunoglobulin; expressing the recovered nucleic acid molecules in a host cell; and recovering antibodies that bind CD38 and CD3, respectively, from the host cell.

[0176] As non-limiting examples, the disclosure provides for the preparation of CD3 binding constructs, where host cells may be co-transfected with nucleic acids encoding a combination of the following: SEQ ID NO:31 and SEQ ID NO:35; SEQ ID NO:31 and SEQ ID NO:36; SEQ ID NO:31 and SEQ ID NO:37; SEQ ID NO:32 and SEQ ID NO:35; SEQ ID NO:32 and SEQ ID NO:36; SEQ ID NO:32 and SEQ ID NO:37; SEQ ID NO:33 and SEQ ID NO:35; SEQ ID NO:33 and SEQ ID NO:36; SEQ ID NO:33 and SEQ ID NO:37; SEQ ID NO:34 and SEQ ID NO:35; SEQ ID NO:34 and SEQ ID NO:36; SEQ ID NO:34 and SEQ ID NO:37; SEQ ID NO:38 and SEQ ID NO:39; or SEQ ID NO:40 and SEQ ID NO:41.

[0177] As non-limiting examples, the disclosure provides CD38 binding constructs, wherein a host cell is capable of expressing any of the following: SEQ ID NO:2 and SEQ ID NO:3; SEQ ID NO:4 alone; SEQ ID NO:5 alone; SEQ ID NO:6 alone; SEQ ID NO:7 and SEQ ID NO:12; SEQ ID NO:7 and SEQ ID NO:13; SEQ ID NO:7 and SEQ ID NO:14; SEQ ID NO:7 and SEQ ID NO:15; SEQ ID NO:7 and SEQ ID NO:16; SEQ ID NO:31 and SEQ ID NO:37; SEQ ID NO:8 and SEQ ID NO:12; SEQ ID NO:8 and SEQ ID NO:13; SEQ ID NO:8 and SEQ ID NO:14; SEQ ID NO:8 and SEQ ID NO:15; SEQ ID NO:8 and SEQ ID NO:16; The CD38 VHO arm may be co-transfected with a nucleic acid encoding SEQ ID NO:9 and SEQ ID NO:12; SEQ ID NO:9 and SEQ ID NO:13; SEQ ID NO:9 and SEQ ID NO:14; SEQ ID NO:9 and SEQ ID NO:15; SEQ ID NO:9 and SEQ ID NO:16; SEQ ID NO:10 and SEQ ID NO:12; SEQ ID NO:10 and SEQ ID NO:13; SEQ ID NO:10 and SEQ ID NO:14; SEQ ID NO:10 and SEQ ID NO:15; SEQ ID NO:10 and SEQ ID NO:16; SEQ ID NO:11 and SEQ ID NO:12; SEQ ID NO:11 and SEQ ID NO:13; SEQ ID NO:11 and SEQ ID NO:14; SEQ ID NO:11 and SEQ ID NO:15; or SEQ ID NO:11 and SEQ ID NO:16. The CD38 VHO arm may be expressed alone as any one of SEQ ID NOs:17-30. The CD38 binding arm may have a tandem sequence consisting of any pair of sequences selected from SEQ ID NO:4-linker-SEQ ID NO:5; SEQ ID NO:4-linker-SEQ ID NO:6, SEQ ID NO:5-linker-SEQ ID NO:4, SEQ ID NO:5-linker-SEQ ID NO:6, SEQ ID NO:6 linker-SEQ ID NO:4, SEQ ID NO:6-linker-SEQ ID NO:5, and SEQ ID NOs:17-30.

[0178] Some embodiments provide a bispecific antibody comprising an antibody sequence targeting CD3 selected from a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NOs: 31-34; and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NOs: 35-40; and an antibody sequence targeting CD38 selected from the amino acid sequences set forth in SEQ ID NOs: 2-30. Fc regions of anti-CD38, anti-CD3, and CD3xCD38 bispecific antibodies

[0179] The anti-CD38, anti-CD3, and CD3xCD38 bispecific antibodies disclosed herein may comprise an altered Fc region, which comprises at least one amino acid modification relative to the native Fc region, e.g., to extend the half-life of the bispecific antibody, to increase the resistance of the bispecific antibody to proteolysis, to reduce effector functionality of the bispecific antibody, to facilitate generation of the bispecific antibody by Fc heterodimerization, to facilitate multimerization of the bispecific antibody, and / or to improve manufacturing and drug stability of the bispecific antibody.

[0180] In some embodiments, the Fc domain is altered to allow for silencing of the Fc domain to minimize effector function activity that can lead to immune cell depletion and cytokine release syndrome.

[0181] In some embodiments, the anti-CD38, anti-CD3, and CD3xCD38 bispecific antibodies described herein comprise an altered Fc region, where the naturally occurring Fc region has been altered to increase the half-life of the antibody in a biological environment compared to the naturally occurring parent antibody, e.g., serum half-life or half-life as measured by an in vitro assay. Exemplary mutations that can be made alone or in combination are T250Q, M252Y, I253A, S254T, T256E, P257I, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A, and H435R mutations.

[0182] In certain embodiments, the half-life extension is c This can be achieved by engineering the M252Y / S254T / T256E mutations in , where residue numbering is according to the EU index (Dall'Acqua, Kiener et al. 2006).

[0183] In certain embodiments, the half-life extension is cThis can also be achieved by engineering the M428L / N434S mutation in (Zalevsky, Chamberlain et al. 2010).

[0184] In certain embodiments, the half-life extension is c This can also be achieved by engineering the T250Q / M428L mutation in (Hinton, Xiong et al. 2006).

[0185] In certain embodiments, the half-life extension is c This can also be achieved by engineering the N434A mutation in (Shields, Namenuk et al. 2001).

[0186] In certain embodiments, the half-life extension is c This can also be achieved by engineering the T307A / E380A / N434A mutations in (Petkova, Akilesh et al. 2006).

[0187] F in antibody half-life extension c The effect of the manipulation was compared with that of natural IgG F c The antibody can be evaluated in a PK study in mice in comparison to an antibody having the

[0188] In some embodiments, the anti-CD38, anti-CD3, and CD3xCD38 bispecific antibodies described herein comprise an altered Fc region, wherein the naturally occurring F c The region has been altered to enhance the antibody resistance to proteolysis by proteases that cleave the wild-type antibody between or at residues 222-237 (EU numbering).

[0189] In certain embodiments, resistance to proteolysis can be achieved by engineering E233P / L234A / L235A mutations and a G236 deletion in the hinge region compared to the native parent antibody, with residue numbering according to the EU index (Kinder, Greenplate et al. 2013).

[0190] When effector functionality is not desired, the antibodies of the present disclosure may be modified to include activated F c γ receptor (F c γR) and / or inhibit F-dependent cytotoxicity, such as C1q binding, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) or phagocytosis (ADCP). c At least one mutation that reduces effector function was present in antibody F c can be further manipulated to introduce

[0191] activation F c F can be mutated to reduce antibody binding to γR and subsequently reduce effector function. c The positions are those described, for example, in (Xu, Alegre et al. 2000) (Vafa, Gilliland et al. 2014) (Bolt, Routledge et al. 1993, Shields, Namenuk et al. 2001, Chu, Vostiar et al. 2008). c F-mediated cell activation cMutations have also been described as sigma mutations for IgG1, IgG2 and IgG4 (Tam, McCarthy et al. 2017). Exemplary mutations that can be made alone or in combination are K214T, E233P, L234V, L234A, G236 deletion, V234A, F234A, L235A, G237A, P238A, P238S, D265A, S267E, H268A, H268Q, Q268A, N297A, A327Q, P329A, D270A, Q295A, V309L, A327S, L328F, A330S and P331S mutations in IgG1, IgG2, IgG3 or IgG4.

[0192] Exemplary combination mutations that can be made to reduce ADCC include L234A / L235A in IgG1, V234A / G237A / P238S / H268A / V309L / A330S / P331S in IgG2, F234A / L235A in IgG4, S228P / F234A / L235A in IgG4, N297A in IgG1, IgG2, IgG3 or IgG4, V234A / G237A in IgG2, K214T / E233P / L234V / L235A / G236 deletion in IgG1. F234A / L235A / G237A / P238S / H268A / A330S / P331S in IgG1; S228P / F234A / L235A / G237A / P238S in IgG4; and S228P / F234A / L235A / G236 deletion / G237A / P238S in IgG4. c Hybrid IgG 2 / 4 F c You can also use a domain.

[0193] In some embodiments, the CD3xCD38 bispecific antibody comprises an altered Fc region, where the native Fc region has been altered to promote the generation of the bispecific antibody by Fc heterodimerization.

[0194] In certain embodiments, Fc heterodimerization can be achieved by engineering F405L and K409R mutations in two parent antibodies and generating bispecific antibodies in a process known as Fab arm exchange (Labrijn, Meesters et al. 2014).

[0195] In certain embodiments, Fc heterodimerization can also be achieved by Fc mutations to facilitate a knob-in-hole strategy (see, for example, International Publication WO 2006 / 028936). An amino acid with a small side chain (hole) is introduced into one Fc domain, and an amino acid with a large side chain (knob) is introduced into the other Fc domain. After co-expression of the two heavy chains, a heterodimer forms as a result of preferential interaction between the heavy chain with the "hole" and the heavy chain with the "knob" (Ridgway, Presta et al. 1996). Exemplary Fc mutation pairs that form knobs and holes are: T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S and T366W / T366S / L368A / Y407V.

[0196] In certain embodiments, Fc heterodimerization can also be achieved by Fc mutations to promote electrostatic match interaction strategies (Gunasekaran, Pentony et al. 2010). Mutations can be engineered to generate positively charged residues in one Fc domain and negatively charged residues in the other Fc domain, as described in US Patent Publication US2010 / 0015133; US Patent Publication US2009 / 0182127; US Patent Publication US2010 / 028637 or US Patent Publication US2011 / 0123532. Heavy chain heterodimerization can be formed by electrostatic match interaction between two mutated Fcs.

[0197] In some embodiments, the CD3×CD38 bispecific antibody comprises the modified F c domain, where bispecific antibodies normally exist as monomers in serum and solution, Naturally occurring Fc regions are modified to facilitate antibody multimerization upon interaction with cell surface receptors. c Mutations include, but are not limited to, the E345R mutation, the E430G mutation, the E345R / E430G mutation, the E345R / E430G / Y440R mutation as described in (Diebolder, Beurskens et al. 2014). Such mutations also include, but are not limited to, the T437R mutation, the T437R / K248E mutation, and the T437R / K338A mutation as described in (Zhang, Armstrong et al. 2017). antibody modification

[0198] Antibodies further comprising conservative modifications are within the scope of this disclosure. "Conservative modifications" refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Conservative modifications include amino acid substitutions, additions, and deletions. Conservative substitutions are those in which an amino acid is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been well defined and include amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amides (e.g., asparagine, glutamine), beta-branched side chains (e.g., threonine, valine, isoleucine) and sulfur-containing side chains (cysteine, methionine). Additionally, any naturally occurring residue in the polypeptide can be substituted with alanine, as previously described for alanine scanning mutagenesis. Amino acid substitutions for the antibodies of the present disclosure can be made by known methods, for example, PCR mutagenesis (U.S. Pat. No. 4,683,195). Alternatively, for example, a library of variants can be made using random (NNK) or non-random codons, for example, the DVK codons that code for 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp). The resulting antibody variants can be evaluated for their characteristics using the assays described herein.

[0199] The antibodies of this disclosure can be post-translationally modified by processes such as glycosylation, isomerization, deglycosylation, or non-naturally occurring covalent modifications, such as the addition of polyethylene glycol moieties (PEGylation) and lipidation. Such modifications can occur in vivo or in vitro. For example, the antibodies of this disclosure can be conjugated to polyethylene glycol (PEGylation) to improve their pharmacokinetic profile. Conjugation can be performed by techniques known to those skilled in the art. Conjugation of therapeutic antibodies with PEG has been shown to enhance pharmacodynamics without interfering with function.

[0200] The antibodies of the present disclosure can be modified to improve stability, selectivity, cross-reactivity, affinity, immunogenicity, or other desirable biological or biophysical properties, and this is within the scope of the present disclosure. Antibody stability is influenced by several factors, including (1) the core packing of individual domains, which affects its intrinsic stability; (2) protein / protein interface interactions, which affect HC and LC pairing; (3) burial of polar and charged residues; (4) H-bond networks for polar and charged residues; and (5) surface charge and polar residue distribution, among other intra- and intermolecular forces (Worn and Pluckthun 2001). Potential structure-destabilizing residues can be identified based on the crystal structure of the antibody or, in certain cases, by molecular modeling, and the effect of the residues on antibody stability can be assessed by making and evaluating variants with mutations at the identified residues. One way to increase antibody stability is to increase the thermal transition midpoint (T m In general, protein T m It correlates with its stability and inversely correlates with its susceptibility to unfolding and denaturation and degradation processes in solution, which depends on the tendency of the protein to unfold. Several studies have found a correlation between the ranking of the physical stability of formulations, measured as thermal stability by DSC, and physical stability measured by other methods. Formulation studies have shown that Fab T mThis suggests that the variability in the mAb response may have implications for the long-term physical stability of the corresponding mAbs.

[0201] The antibodies of the disclosure contain amino acid substitutions that improve manufacturing and drug stability. c An example for IgG1 is H224S (or H224Q) in the hinge 221-DKTHTC-226 (Eu numbering), which blocks radical-induced cleavage; for IgG4, the S228P mutation blocks half-antibody exchange. Shielding or Masking Domains

[0202] As non-limiting examples, the present disclosure provides bispecific antibodies comprising a shielding domain (also referred to as a masking domain, mask, or cap) selected from a shielding domain amino acid sequence set forth in SEQ ID NOs: 42-45 of Table 5 capable of shielding binding of CD38 Fab, and a shielding domain amino acid sequence set forth in SEQ ID NOs: 46-52 of Table 5 capable of shielding binding of CD3 Fab. Some embodiments provide various shielding or caps that mask CD38 binding as set forth in SEQ ID NOs: 42-51. Some embodiments provide various shielding or caps that mask CD3 binding as set forth in SEQ ID NOs: 46-52. Table 5 [Table 5-1] [Table 5-2] Protease-cleavable linkers

[0203] A protease-cleavable linker comprises a protease-cleavable peptide substrate that links the shielding domain to an antibody heavy or light chain. A protease-cleavable linker comprises one or more protease substrate sequences and an optional linker spacer sequence (see Figures 5-7). In some embodiments, the shielding sequence is present as a combination of sequences that can be fused to the heavy and light chains. For example, for each of the two Fab arm domains of a bispecific antibody, the shielding sequence is fused to the N-terminus of the antibody heavy chain via one protease-cleavable linker, and the complement sequence is fused to the N-terminus of the antibody light chain via another protease-cleavable linker.

[0204] Many diseased tissues, including tumor microenvironments and inflammatory sites, have large amounts of various types of proteases whose overexpression correlates with disease progression. In diseased tissues, the protease-cleavable linker sequence of the shielding antibody can be recognized by the appropriate type of protease, and the shield can be cleaved off from the antibody chain. For example, in one binding arm of a shielded bispecific antibody, the protease may cleave both of the two protease-cleavable linkers or cleave one of the two protease-cleavable linker sequences, so that the shielding domain is inactive. In either case, the shielding domain will not be able to interfere with or block the binding of the Fab arm to its target antigen. As a result, the shielding antibody is converted into an active antibody, binds to its target, and exerts functional activity (Figure 7).

[0205] In some embodiments, the protease-cleavable linker sequences linking the two shielding domains to the two Fab domains of the shield antibody comprise the same sequence in order to be cleaved by the same type of protease.

[0206] In some embodiments, the protease-cleavable linker sequence connecting the two masking domains and the two Fab domains of the shield antibody comprises different sequences having substrate sequences that are cleaved by different types of proteases.

[0207] Among the family of matrix metalloproteinases (MMPs), MMP2 and MMP9 are upregulated in many types of cancer, including breast cancer, colorectal cancer, and lung cancer. Moreover, the expression and activity of MMP2 and MMP9 are also associated with the progression of many autoimmune and inflammatory diseases, including rheumatoid arthritis, psoriasis, multiple sclerosis, chronic obstructive respiratory disease, inflammatory bowel disease, and osteoporosis (Lin, Lu et al. 2020). The present disclosure provides protease-cleavable linker sequences that include substrate peptide sequences that are cleaved by MMP2 and MMP9. As a non-limiting example, the present disclosure provides substrate peptide sequences that are cleavable by MMP2 and MMP9, as set forth in SEQ ID NOs: 53-57. As a non-limiting example, the present disclosure provides a substrate peptide sequence that is cleavable by MMP3, as set forth in SEQ ID NO: 58.

[0208] Urokinase plasminogen activator (uPA) has been reported to be overexpressed in many types of cancer, especially breast cancer (Banys-Paluchowski, Witzel et al. 2019). uPA is a serine protease that can catalyze the conversion of plasminogen to plasmin, which can degrade basement membranes or extracellular matrix. Matrix degradation facilitates tumor cell migration and invasion into surrounding tissues. The present disclosure provides protease-cleavable linker sequences that include substrate peptide sequences that are cleaved by uPA. As a non-limiting example, the present disclosure provides uPA-cleavable substrate peptide sequences set forth in Table 6 as SEQ ID NOs: 59 and 60. Table 6 [Table 6]

[0209] Protease-cleavable linkers of the disclosure can include, for example, one or more linker peptides inserted between the shielding sequence and the protease substrate peptide sequence and / or between the protease substrate peptide sequence and the antibody Fab.

[0210] Suitable linkers (also referred to as "spacers") are readily selected and can be of any suitable length, from 1 amino acid to 30 amino acids (e.g., any particular integer between 1 and 30), or from 1 amino acid (e.g., Gly) to about 20 amino acids, 2 to 15, 3 to 12, 4 to 10, 5 to 9, 6 to 8, or 7 to 8 amino acids.

[0211] Exemplary linkers include glycine polymers (G) n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n , and (GGGS) n {wherein n is at least one integer, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20}, glycine-alanine polymers, alanine-serine polymers, alanine-proline, immunoglobulin isotype and subtype hinges including IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, and other flexible linkers known in the art. Both Gly and Ser are unstructured and therefore can serve as neutral tethers between moieties.

[0212] In certain embodiments, the linker is a glycine polymer. Glycine has access to significantly more phi-psi space than alanine and is less restricted than residues with longer side chains (Scheraga 2008). Exemplary linkers can include amino acid sequences containing, but are not limited to, GGS; GGSG; GGSGG; GGGGS; GGSSG; GGGSG; GGSSG; GSSSG, and the like.

[0213] In certain embodiments, the linker is an alanine-proline polymer. Exemplary linkers include, but are not limited to, (AP) n wherein n is at least one integer, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0214] In certain embodiments, the linker is a rigid linker (Chen, Zaro et al. 2013). An exemplary rigid linker is a proline-rich sequence, (XP), with X designating any amino acid, preferably Ala, Lys, or Glu. n Exemplary rigid linkers include amino acid sequences including, but not limited to, (EAAAK) n The amino acid sequences may also include, but are not limited to, an alpha-helix-forming linker having the sequence: Antibody expression and purification

[0215] The antibodies of the disclosure may be encoded by one or more nucleic acids for protein expression. For example, a shielded CD3xCD38 bispecific antibody of the disclosure may be encoded by a single nucleic acid (e.g., a single nucleic acid comprising nucleotide sequences encoding the light and heavy chain polypeptides of the shielded antibody) or by two or more separate nucleic acids, each of which encodes a different portion of the shielded parent antibody.

[0216] The appropriate recombinant DNA is prepared by recombinant DNA techniques, then transfected into mammalian cells and the corresponding anti-CD3 and anti-CD38 antibodies are expressed, purified, identified and / or screened.

[0217] Bispecific antibodies are generated from anti-CD3 and anti-CD38 antibodies using controlled Fab arm exchange or other bispecific antibody manufacturing processes that result in bispecific antibodies that exhibit the biological effect of simultaneous binding to CD38 and CD3. The affinity and inhibitory efficiency of the bispecific antibodies are identified by in vitro experiments.

[0218] The nucleic acids described herein can be inserted into vectors, e.g., nucleic acid expression vectors and / or targeting vectors. Such vectors can be used in various ways, e.g., for expression of a pro-antibody with a masking domain (shielded antibody) described herein in a cell or transgenic animal. The vector is typically selected to be functional in the host cell in which the vector will be used. The nucleic acid molecule encoding the antibody, e.g., a pro-antibody with a masking domain described herein, can be amplified / expressed in prokaryotic, yeast, insect (baculovirus system) and / or eukaryotic host cells. The choice of host cell will depend in part on whether the antibody disclosed herein, such as the shielded CD3×CD38 bispecific antibody described herein, is to be post-translationally modified (e.g., glycosylated and / or phosphorylated). If so, yeast, insect or mammalian host cells are preferred. Expression vectors typically contain one or more of the following components: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a leader sequence for secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding a polypeptide to be expressed, and a selectable marker element.

[0219] In some embodiments, a leader or signal sequence is engineered at the N-terminus of an antibody, such as the shielded CD3xCD38 bispecific antibody described herein, to direct its secretion. Secretion of the shielded CD3xCD38 bispecific antibody from a host cell will result in removal of the signal peptide from the antibody. Thus, the mature shielded CD3xCD38 bispecific antibody will lack any leader or signal sequence. In some embodiments, such as when glycosylation is desired in a eukaryotic host cell expression system, various presequences can be engineered to improve glycosylation or yield. For example, the peptidase cleavage site of the signal peptide can be altered or a prosequence can be added, which can affect glycosylation.

[0220] The present disclosure further provides a cell (e.g., an isolated or purified cell) comprising a nucleic acid or vector of the present disclosure. The cell can be any type of cell that can be transformed with a nucleic acid or vector of the present disclosure that can produce the encoded polypeptide. For example, to express the shielded CD3xCD38 bispecific antibody described herein, DNA encoding the partial or full-length light and heavy chains obtained as described above is inserted into an expression vector such that the genes are operably linked to transcriptional and translational control sequences.

[0221] Methods for introducing nucleic acids and vectors into isolated cells, and for culturing and selecting transformed host cells in vitro, are known in the art and include the use of calcium chloride-mediated transformation, transduction, conjugation, triparental mating, DEAE, dextran-mediated transfection, infection, membrane fusion with liposomes, high-velocity bombardment with DNA-coated microprojectiles, direct microinjection into single cells, and electroporation.

[0222] After introducing the nucleic acid or vector of this disclosure into the host cell, the cells are cultured under conditions suitable for expression of the encoded sequence. The antibody, antigen-binding fragment, or portion of the antibody can then be isolated from the cells.

[0223] In certain embodiments, two or more vectors that together encode the shield CD3xCD38 bispecific antibodies described herein can be introduced into a host cell.

[0224] Purification of the antibodies described herein, e.g., shielded CD3xCD38 bispecific antibodies, secreted into the cell culture medium can be accomplished using a variety of techniques, including affinity, immunoaffinity or ion exchange chromatography, molecular sieve chromatography, preparative gel electrophoresis or isoelectric focusing, chromatofocusing, and high pressure liquid chromatography. c The antibody containing the region is F c The antibody can be purified by affinity chromatography with Protein A, which selectively binds to the Shielded CD3xCD38 bispecific antibody. Modified forms of antibodies, such as the shielded CD3xCD38 bispecific antibody, can be prepared with affinity tags, such as hexahistidine or other small peptides, such as FLAG (Eastman Kodak Co., New Haven, Conn.) or Myc (Invitrogen), at either their carboxyl or amino termini and purified by a one-step affinity column. For example, since polyhistidine binds to nickel with excellent affinity and specificity, a nickel affinity column (such as a Qiagen® nickel column) can be used for purification of polyhistidine-tagged selective binding agents. In some instances, two or more purification steps can be used. Effect of SHIELD CD3 x CD38 bispecific antibody on binding and functional activity

[0225] The shielded CD3xCD38 bispecific antibody disclosed herein may inhibit or block the ability of the Fab arms to bind to their respective antigens, CD3 and CD38. The masking domain may reduce the maximum binding capacity of the shielded bispecific antibody when binding to its respective antigen. The masking domain may also reduce the binding affinity of the shielded bispecific antibody when binding to its respective antigen.

[0226] When the masking domain is cleaved off by a protease, the shielded antibody is converted into an active bispecific antibody with recovery of the antibody's ability to bind to its antigen. Removal of the masking domain from the shielded bispecific antibody is achieved in an in vitro protease cleavage assay using recombinant or purified proteases. Removal of the masking domain from the shielded bispecific antibody is also achieved in vivo by overexpressed proteases at the disease site. Removal of the masking domain is assessed by comparing the molecular weight of the heavy and light chains of the shielded antibody with the masking domain to the active antibody without the masking domain by SDS-PAGE, IEX, or HIC analysis.

[0227] In vitro and cell-based assays are well described in the art for use in measuring pro-antibodies (shielded bispecifics), active antibodies, and converted antibodies after protease cleavage in binding to their antigen. For example, antibody binding may be measured by ELISA by immobilizing recombinant or purified antigen, sequestering the antibody with the immobilized antigen, and measuring the amount of bound antibody. This can also be performed using a Biacore® instrument for kinetic analysis of binding interactions. For cell-based binding assays, antibody binding may be measured by flow cytometry by incubating the antibody with cells expressing the antigen on their cell surface and measuring the amount of antibody bound to the cell surface antigen. Pharmaceutical Compositions

[0228] Antibodies, such as the shield CD3xCD38 bispecific antibody for use according to the present disclosure, can be formulated in compositions, particularly pharmaceutical compositions, for use in the methods herein. Such compositions comprise a therapeutically or prophylactically effective amount of an antibody, e.g., a bispecific antibody, as described in this disclosure, in a mixture with a suitable carrier, e.g., a pharma- ceutically acceptable agent, and a suitable carrier. Typically, the antibodies described in this disclosure are sufficiently purified for administration to animals prior to formulation in a pharmaceutical composition.

[0229] In some embodiments, the disclosure provides a composition comprising an anti-CD38 antibody or antigen-binding portion thereof, hi some embodiments, the disclosure provides a pharmaceutical composition comprising an anti-CD38 antibody or antigen-binding portion thereof and a pharma- ceutical acceptable carrier.

[0230] In some embodiments, the disclosure provides a composition comprising an anti-CD3 antibody or antigen-binding portion thereof. In some embodiments, the disclosure provides a pharmaceutical composition comprising an anti-CD3 antibody or antigen-binding portion thereof and a pharma- ceutical acceptable carrier.

[0231] In some embodiments, the disclosure provides compositions comprising a shielded or unshielded CD3xCD38 bispecific antibody, hi some embodiments, the disclosure provides pharmaceutical compositions comprising a shielded or unshielded CD3xCD38 bispecific antibody and a pharma- ceutical acceptable carrier.

[0232] Pharmaceutically acceptable agents include carriers, excipients, diluents, antioxidants, preservatives, colorants, flavorings and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, isotonicity agents, co-solvents, wetting agents, complexing agents, buffering agents, antimicrobial agents and surfactants.

[0233] The compositions may be in liquid form or in lyophilized or freeze-dried form and may include one or more cryoprotectants, excipients, surfactants, high molecular weight structural additives and / or bulking agents.

[0234] In some embodiments, the composition comprises a shielded CD3xCD38 bispecific antibody, or an anti-CD38 antibody or antigen-binding portion thereof, or an anti-CD3 antibody or antigen-binding portion thereof, and at least one buffer, at least one stabilizer, and / or at least one detergent.

[0235] In some embodiments, the compositions disclosed herein are liquid. In some embodiments, the compositions are formulated for subcutaneous injection. In some embodiments, the compositions are sterile. In some embodiments, the compositions further comprise histidine HCl, trehalose, methionine, and / or polysorbate.

[0236] The composition may be suitable for parenteral administration. Exemplary compositions are suitable for injection or infusion into animals by any route available to those skilled in the art, including intra-articular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intracemall), intraventricular, intramuscular, intraocular, intraarterial, intralesional, intrarectal, transdermal, oral and inhalation routes.

[0237] The pharmaceutical compositions described herein can be formulated for controlled or sustained delivery in a manner that provides local concentration (e.g., bolus, depot effect, topical application), sustained release and / or increased stability or half-life of the product in a particular local environment.

[0238] In some embodiments, the CD3xCD38 bispecific antibody, anti-CD38 antibody or antigen-binding portion thereof, or anti-CD3 antibody or antigen-binding portion thereof may be present in the pharmaceutical composition at a concentration of 1 mg / mL to 250 mg / mL, 10 mg / mL to 250 mg / mL, 1 mg / mL to 100 mg / mL, 2 mg / mL to 50 mg / mL, and 2 mg / mL to 40 mg / mL. Methods of treatment and use

[0239] In some embodiments, the present disclosure provides a method of treating or preventing a disease or disorder in a subject in need of such treatment or prevention, comprising administering to the subject an effective amount of a shielded CD3xCD38 bispecific antibody, or an anti-CD38 antibody, or antigen-binding portion thereof, disclosed herein. The disease or disorder is selected from CD38 mediated diseases or disorders, e.g., human cancers including gastric and colorectal cancer, pancreatic cancer, prostate cancer, lung cancer, hepatocellular carcinoma, triple negative breast cancer, nasopharyngeal cancer, cervical cancer, hematological malignancies (such as MM, lymphoma, acute myeloid leukemia, chronic lymphocytic leukemia, acute B lymphoblastic leukemia and others), heart disease, viral infections including HIV infection, asthma and other respiratory inflammatory diseases, allergic airway diseases, maternal tolerance, autism spectrum disorders, glomerulosclerosis, inflammatory bowel disease, rheumatoid arthritis, diabetes mellitus, chronic autoimmune thyroiditis and age-related neurodegenerative and neuroinflammatory diseases such as Graves' disease, Alzheimer's disease. In some embodiments, the CD38-mediated disease or disorder is selected from human cancers, including gastric and colorectal cancer, pancreatic cancer, prostate cancer, lung cancer, hepatocellular carcinoma, triple-negative breast cancer, nasopharyngeal cancer, cervical cancer, and hematological malignancies.

[0240] Some embodiments also provide the use of a CD3xCD38 bispecific antibody, or an anti-CD38 antibody or an antigen-binding portion thereof, in the manufacture of a medicament for the treatment or prevention of cancer, preferably lung cancer, breast cancer, colorectal cancer, gastric cancer, intestinal cancer, and / or pancreatic cancer.

[0241] In some embodiments, the present disclosure provides a shielded CD3xCD38 bispecific antibody herein, or an anti-CD38 antibody or antigen-binding portion thereof disclosed herein, for use in treating or preventing a disease or disorder selected from a CD38-mediated disease or disorder.

[0242] In some embodiments, the present disclosure provides the use of a shielded CD3xCD38 bispecific antibody, or an anti-CD38 antibody, or antigen-binding portion thereof, disclosed herein, for the manufacture of a medicament for use in treating or preventing a disease or disorder selected from a CD38-mediated disease or disorder.

[0243] In some embodiments, the present disclosure provides a shielded CD3xCD38 bispecific antibody, or an anti-CD38 antibody or antigen-binding portion thereof, as described herein, for use in treating or preventing gastric cancer, lung cancer, pancreatic cancer, colorectal cancer, and other cancers. In contrast to the corresponding therapeutic antibodies, the shielded CD3xCD38 bispecific antibodies may have comparable efficacy in treating these diseases due to the conversion of the shielded antibody into an active antibody specific to the disease site by removal of the shielding domain by proteases overexpressed at the disease site. However, the shielded antibody may have reduced systemic toxicity due to masking of the antibody activity by the shielding domain in normal tissues that lack sufficient amounts of proteases required for trimming off the masking domain. The shielded bispecific antibodies described herein may be as effective in treating diseases as the corresponding therapeutic antibodies, but with a much improved safety profile. Due to the improved safety profile, high levels of a dose containing the shielded bispecific antibody may be administered to patients with improved therapeutic efficacy.

[0244] In some embodiments, the disclosure also provides a method of treating cancer in a subject, comprising administering a therapeutically effective amount of a shielded CD3xCD38 bispecific antibody. The disclosure also provides the use of the shielded bispecifics provided herein in methods of treating cancer, and the use of the shielded CD3xCD38 bispecific antibodies provided herein in the manufacture of a medicament for use in cancer. Exemplary cancers include, but are not limited to, MM, non-small cell lung cancer, acute myeloid leukemia, female breast cancer, pancreatic cancer, colorectal cancer, and peritoneal cancer.

[0245] In some embodiments, the bispecific antibodies disclosed herein, or anti-CD38 antibodies, or antigen-binding portions thereof, effectively inhibit the association of the CD38 receptor with integrins and / or downstream signaling.

[0246] In some embodiments, the bispecific antibodies disclosed herein, or anti-CD38 antibodies or antigen-binding portions thereof, can be used in combination with chemotherapy. For example, a combination regimen for treating cancer can use higher doses of chemotherapy and a CD3xCD38 bispecific antibody to determine the best synergistic partner.

[0247] In some embodiments, the present disclosure provides a conjugate of a bispecific antibody disclosed herein, or an anti-CD38 antibody, or antigen-binding portion thereof, with another therapeutic agent. In some embodiments, the other therapeutic agent is a cytotoxin or a radioisotope.

[0248] Unless otherwise indicated herein or otherwise clearly contradicted by context, all combinations of the various elements described herein are within the scope of the disclosure.

[0249] This disclosure will be better understood from the following illustrative details, however, those skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the present disclosure, which is more fully described in the claims that follow thereafter. EXAMPLES

[0250] Example 1: Expression and purification of anti-CD38 and anti-CD3 antibodies Masked and unmasked anti-CD3 and anti-CD38 antibody genes were placed in separate plasmids to evaluate the preparation of monoclonal anti-CD3 and anti-CD38 antibodies, as well as anti-CD3 and anti-CD38 bispecific antibodies. For shielded antibodies, heavy and light chain constructs expressing anti-CD3 and anti-CD38 shielded mAbs were prepared. Plasmids encoding the heavy and light chains of these anti-CD3 and anti-CD38 masked antibodies were co-transfected into Expi293F cells according to the transfection kit instructions (Thermo Scientific). Cells were spun down 5 days after transfection, and the supernatant was passed through a 0.2 μm filter. Purification of the expressed masked antibody supernatant was performed by affinity chromatography on a Protein A agarose column (GE Healthcare Life Sciences). The purified masked antibodies were buffer exchanged into DPBS, pH 7.2 by dialysis, and the protein concentration was measured by UV absorbance at 280 nm. Unshielded anti-CD3 and anti-CD38 monoclonal antibodies were similarly prepared and purified.

[0251] Anti-CD3 and anti-CD38 shielded mAbs had masking domains fused to the N-terminus of the antibody heavy and light chains with substrate sequences for MMP2 / MMP9. Purified masked antibodies were characterized by SDS-PAGE analysis.

[0252] Example 2: Protease Digestion of Antibodies with Masking Domains

[0253] An in vitro protease cleavage assay was designed to assess whether the shielding domain is removed from the masked Ab by proteases. Recombinant human MMP2 was activated by incubation with p-aminophenylmercuric acetate (APMA) according to the manufacturer's instructions (R&D Systems). 10 mg of masked Ab was incubated with 50 ng of activated MMP2 overnight at 37°C. Digestion of masked mAb was assessed by SDS-PAGE under reducing conditions. The molecular weights of the heavy and light chains of digested masked Ab were slightly smaller than those of the corresponding undigested parent antibody. After protease treatment, the molecular weight of the capped mAb was close to that of the reference mAb.

[0254] Example 3: Anti-CD3 antibodies bind to recombinant human and cynomolgus CD3 antigens in ELISA assays

[0255] Biotin-labeled human recombinant CD3 protein, consisting of CD3ε and CD3δ chains, was bound to a streptavidin-coated ELISA plate to capture the antigen on the solid phase. Different concentrations of test anti-CD3 antibodies were then added to the coated plate, and bound antibodies were detected and quantified using standard ELISA protocols. The results shown in Figure 8A demonstrate the ability of these anti-CD3 antibodies to bind to the intended human CD3 target. The antibodies show a range of affinities for the antigen in this assay format. This range of affinities is valuable, as different CD3 affinities may be ideal for disease-specific applications or specific combinations with other antibodies in bispecific formats.

[0256] Biotin-labeled cynomolgus monkey recombinant CD3 protein, consisting of CD3ε and CD3δ chains, was bound to streptavidin-coated ELISA plates to capture the antigen on the solid phase. Different concentrations of test anti-CD3 antibodies were then added to the coated plates, and bound antibodies were detected and quantified using standard ELISA protocols. The results shown in FIG. 8B demonstrated the ability of this anti-CD3 species to bind and cross-react with cynomolgus monkey CD3 protein, in addition to their demonstrated binding to human CD3 protein. The ability to also bind to cynomolgus monkey CD3 protein is an important functionalization property for drug development, as it facilitates the use of these animals for toxicology and other necessary investigations on these antibodies and especially bispecific antibodies that contain them.

[0257] Bispecific antibodies consisting of a single anti-CD38 arm combined with various anti-CD3 arms are mixed with human PBMCs containing CD3 positive T cells at a series of increasing concentrations of the bispecific antibodies. Bispecific antibody binding was detected and quantified using standard flow cytometry methods. The results are shown in FIG. 9. These data demonstrate that the tested anti-CD3 antibodies, in addition to their demonstrated ability to bind isolated recombinant proteins, can bind to human CD3 protein when it is naturally presented on the surface of the intended target T cells. Thus, the CD3 antibodies were demonstrated by both ELISA and cell binding.

[0258] Example 4: Anti-CD38 antibodies bind to recombinant human, recombinant cynomolgus monkey, and recombinant mouse CD38 antigens in ELISA assays

[0259] Biotin-labeled recombinant CD38 protein was bound to streptavidin-coated ELISA plates to capture the antigen on the solid phase. Different concentrations of test anti-CD38 antibodies were then added to the coated plates, and bound antibodies were detected and quantified using standard ELISA protocols. The results shown in Figure 10 demonstrated the ability of these anti-CD38 species to bind to the intended human CD38 target and, in some cases, to the mouse antigen. Daratumumab is a known and currently commercially available D38-specific antibody with clinical activity (heavy and light chain variable regions with SEQ ID NOs: 2, 3) and single domain antibody variable heavy chains with SEQ ID NOs: 5 and 6 were included as positive controls and for comparison purposes. The results shown in Figure 10 demonstrated the ability of these novel anti-CD38 species, SEQ ID NOs: 4, 17, 18, 20, 5, 21, 24, 6, 29, to bind to the intended human CD38 target. The antibodies showed a range of affinities for the antigen in this assay format. The anti-CD38 antibody of SEQ ID NO: 4 may also bind to the cynomolgus CD38 antigen in an ELISA format (FIG. 10B). The anti-CD38 antibody of SEQ ID NO: 4 may also bind to the mouse CD38 antigen in an ELISA format (FIG. 10C). Biotin-labeled cynomolgus recombinant CD38 protein was bound to a streptavidin-coated ELISA plate to capture the antigen on the solid phase. Different concentrations of the test anti-CD38 antibody were then added to the coated plate, and the bound antibody was detected and quantified using a standard ELISA protocol. The results shown in FIG. 10 demonstrated the ability of these novel anti-CD38 species to bind and cross-react with the cynomolgus CD38 target, in addition to their demonstrated binding to human CD38 protein. The ability to also bind to the cynomolgus CD38 protein is an important functionalizing property for drug development, as it facilitates the use of these animals for toxicology and other necessary investigations on these antibodies and especially bispecific antibodies that contain them.

[0260] An ELISA-based binding assay is used to evaluate the binding of CD3xCD38 bispecific antibodies to CD3 and CD38. In this assay, human CD38 is coated onto a plate, and then CD3xCD38 bispecific antibodies or a mixture of CD3 and CD38 antibodies are added along with recombinant CD3 with a His tag. After washing away non-specific binding, the presence of CD3 is detected by an HRP-conjugated anti-his secondary antibody (BioLegend). CD3xCD38 bispecific antibodies mobilized CD3 in a dose-dependent manner, but not by the mixture of the two parent antibodies. Thus, the bispecific antibodies can simultaneously bind CD3 and CD38.

[0261] Example 5: Anti-CD38xCD3 antibodies bind to and activate human CD3-positive T cell lines in the presence of H929, a CD38-positive target cell line

[0262] Bispecific antibodies, each containing a single anti-CD38 arm combined with various anti-CD3 arms, were mixed with a series of increasing concentrations of the bispecific antibody with two cell lines: a reporter T cell line and a target cell line, H929, a CD38-positive multiple myeloma cell line. The reporter T cell line was a human CD3-positive Jurkat T cell line genetically engineered to produce an easily measured signal when the T cell receptors on their surface bind and activate for killing. The target cell line was the H929 human CD38-positive multiple myeloma cell line. For cancer therapy, we expected that the CD38xCD3 bispecific antibody would bind to CD3 on the attacking killer T cells and CD38 on the target cancer cells. The results, shown in Figure 11, demonstrated concentration-dependent activation of T cells as a consequence of the binding of the bispecific antibody to both CD3 and CD38 proteins. These data demonstrated that the anti-CD3 and anti-CD38 arms tested were able to bind to both proteins when they were presented on the cell surface of the cells. Minimal activation was seen in the presence of the inactive arm of the bispecific antibody. The data further supported the conclusion that binding also mediated the desired activation of T cell pathways necessary for targeted cancer cell killing.

[0263] Example 6: Anti-CD38xCD3 antibodies bind to and activate human CD3-positive T cell lines in the presence of L363, a CD38-positive target cell line

[0264] Bispecific antibodies, each containing a single anti-CD38 arm combined with various anti-CD3 arms, were mixed with a series of increasing concentrations of the bispecific antibody with two cell lines: one a reporter T cell line and the other a target cell line. The reporter T cell line was a human CD3-positive Jurkat T cell line genetically engineered to produce an easily measured signal when the T cell receptors on their surface bind and activate for killing. The target cell line was the L363 human CD38-positive multiple myeloma cell line. For cancer therapy, the CD38xCD3 bispecific antibody was expected to bind to CD3 on the attacking killer T cells and to CD38 on the target cancer cells. The results shown in Figure 12 demonstrated concentration-dependent activation of T cells as a consequence of the binding of the bispecific antibody to both CD3 and CD38 proteins. These data demonstrated that the anti-CD3 and anti-CD38 arms tested were able to bind to both proteins when they were presented on the cell surface of the cells. Minimal activation was seen in the presence of the inactive arm of the bispecific antibody. The data further supported the conclusion that binding also mediated the desired activation of T cell pathways required for targeted cancer cell killing.

[0265] Example 7: Anti-CD38xCD3 antibodies bind to and activate human CD3 positive T cell lines in the presence of RPMI 8226, a CD38 positive target cell line

[0266] Bispecific antibodies, each containing a single anti-CD38 arm combined with various anti-CD3 arms, were mixed with a series of increasing concentrations of the bispecific antibody with two cell lines: one a reporter T cell line and the other a target cell line. The reporter T cell line was a human CD3-positive Jurkat T cell line genetically engineered to produce an easily measured signal when the T cell receptors on their surface bind and activate for killing. The target cell line was the RPMI 8226 human CD38-positive multiple myeloma cell line. For cancer therapy, the CD38xCD3 bispecific antibody was expected to bind to CD3 on the attacking killer T cells and to CD38 on the target cancer cells. The results shown in Figure 13 demonstrated concentration-dependent activation of T cells as a consequence of the binding of the bispecific antibody to both CD3 and CD38 proteins. These data demonstrated that the anti-CD3 and anti-CD38 arms tested were able to bind to both proteins when they were presented on the cell surface of the cells. Minimal activation was seen in the presence of the inactive arm of the bispecific antibody. The data further supported the conclusion that binding also mediated the desired activation of T cell pathways required for targeted cancer cell killing.

[0267] Example 8: Anti-CD38 x CD3 antibodies directed PBMCs to kill H929, a CD38-positive target cell line

[0268] Bispecific antibodies, each containing a single anti-CD38 arm combined with various anti-CD3 arms, were mixed with a series of increasing concentrations of the bispecific antibodies with PBMC lots and target cell lines. The target cell line was the H929 human CD38-positive multiple myeloma cell line. The results shown in FIG. 14 demonstrated concentration-dependent T cell killing as a consequence of the binding of the bispecific antibodies to both CD3 and CD38 proteins. These data demonstrated that in addition to their demonstrated ability to bind isolated recombinant proteins, the tested anti-CD3 and anti-CD38 arms were able to bind both proteins when they were presented on the cell surface of the cells. The data further supported the conclusion that the binding also mediated the desired T cell directed target cancer cell killing. These experiments with two different PBMC donor T cells used as effector cells led to potent PBMC donor T cell killing of H929 cells. The data further supported the conclusion that binding to both CD3 and CD38 proteins also resulted in bispecific antibody-mediated activation of human primary T cells and killing of their target cancer cells.

[0269] Example 9: Anti-CD38 x CD3 antibodies directed PBMCs to kill RPMI 8226, a CD38-positive target cell line

[0270] Bispecific antibodies, each containing a single anti-CD38 arm combined with various anti-CD3 arms, were mixed with a series of increasing concentrations of the bispecific antibodies with PBMC lots and target cell lines. The target cell line was the RPMI 8226 human CD38-positive multiple myeloma cell line. The results shown in FIG. 15 demonstrated concentration-dependent T cell killing as a consequence of bispecific antibody binding to both CD3 and CD38 proteins. These data demonstrated that in addition to their demonstrated ability to bind isolated recombinant proteins, the tested anti-CD3 and anti-CD38 arms were able to bind to both proteins when they were presented on the cell surface of the cells. The data further supported the conclusion that binding to both CD3 and CD38 proteins also mediated the desired T cell directed target cancer cell killing. These experiments with four different PBMC donor T cells used as effector cells led to potent PBMC donor T cell killing of RPMI 8226 cells. The data further supported the conclusion that binding to both CD3 and CD38 proteins also resulted in bispecific antibody-mediated activation of human primary T cells and killing of their target cancer cells.

[0271] Example 10: Anti-CD38 x CD3 antibodies directed PBMCs to kill L363, a CD38-positive target cell line

[0272] Bispecific antibodies, each containing a single anti-CD38 arm combined with various anti-CD3 arms, were mixed with a series of increasing concentrations of the bispecific antibodies with PBMC lots and target cell lines. The target cell line was the L363 human CD38-positive multiple myeloma cell line. The results shown in FIG. 16 demonstrated concentration-dependent T cell killing as a consequence of the binding of the bispecific antibodies to both CD3 and CD38 proteins. These data demonstrated that in addition to their demonstrated ability to bind isolated recombinant proteins, the tested anti-CD3 and anti-CD38 arms were able to bind both proteins when they were presented on the cell surface of the cells. The data further supported the conclusion that the binding also mediated the desired T cell directed target cancer cell killing. These experiments with three different PBMC donor T cells used as effector cells led to potent PBMC donor T cell killing of L363 cells. The data further supported the conclusion that binding to both CD3 and CD38 proteins also resulted in bispecific antibody-mediated activation of human primary T cells and killing of their target cancer cells.

[0273] Example 11: Antitumor effect of CD3×CD38 bispecific antibodies in vivo

[0274] The effect of CD3xCD38 bispecific antibodies in tumor cell killing was evaluated in a mouse tumor xenograft model. A novel anti-CD38 antibody, referred to in SEQ ID NO: 4, was generated as an IgG1Fc fusion and combined into a bispecific antibody with the 40G5 CD3 arm, known to be capable of directing T cell killing, as well as evaluating the ability of SEQ ID NO: 4 to direct T cell killing of CD38 expressing cells. This bispecific antibody was evaluated in the NCI-H929 human multiple myeloma xenograft model, in which human PBMCs containing CD3 positive effector T cells were implanted into immunodeficient mice 1 day after subcutaneous implantation of cancer cells (Figures 17B-17C) or 5 days before subcutaneous implantation of cancer cells (Figures 17D-17E). NCI-H929, a CD38 expressing cell line, was implanted into NCG mice for tumor xenograft establishment. Test molecules were administered intraperitoneally to the mice, and antibody-mediated tumor regression was evaluated (Figure 17A). Since it is understood that the outcome of these models depends widely on the nature of the transplanted T cells from different donors, the experiments were performed using two parallel cohorts of mice, each transplanted with T cells from a different donor. Two different sets of CD3 arms were used to generate the CD38xCD3 bispecific antibody. The control CD3 arm was designated 40G5, which contains a heavy chain variable region, SEQ ID NO: 40, and a light chain variable region, SEQ ID NO: 41. The test anti-CD3 arm was designated SP34 v8, which includes a heavy chain variable region, SEQ ID NO: 39, and a light chain variable region, SEQ ID NO: 38. The results of the experiment, shown in FIG. 17, demonstrated that the CD38×CD3 bispecific antibody, SEQ ID NO: 4 in combination with the 40G5 arm and SEQ ID NO: 4 in combination with the SP34 v8 arm, were highly active in inhibiting tumor growth in both cohorts, with partial tumor inhibition in one T cell donor and complete tumor growth inhibition in the second donor. This inhibition was particularly noteworthy because each cohort was also treated with daratumumab, an anti-CD38 antibody approved for clinical use in multiple myeloma and known to be highly active in multiple myeloma patients. The results showed that daratumumab was inactive in this model, while the CD38×CD3 antibody showed clear anti-tumor activity in both donor T cell cohorts. The most important conclusion is that the anti-CD38 arm of the bispecific antibody tested in this study is highly effective against multiple myeloma in vivo. References Akther, S., N. Korshnova, J. Zhong, M. Liang, SM Cherepanov, O. Lopatina, YK Komleva, AB Salmina, T. Nishimura, AA Fakhrul, H. Hirai, I. Kato, Y. Yamamoto, S. Takasawa, H. Okamoto and H. Higashida (2013). "CD38 in the nucleus accumbens and oxytocin are related to paternal behavior in mice." Mol Brain 6: 41. Allard, B., PA Beavis, PK Darcy and J. Stagg (2016). "Immunosuppressive activities of adenosine in cancer." Curr Opin Pharmacol 29: 7-16. Amgen. (2021). "Bincyto." Antonelli, A., G. Baj, P. Marchetti, P. Fallahi, N. Surico, C. Pupilli, F. Malavasi and E. Ferrannini (2001). "Human anti-CD38 autoantibodies raise intracellular calcium and stimulate insulin release in human pancreatic islets." Diabetes 50(5): 985-991. Antonelli, A., P. Fallahi, C. Nesti, C. Pupilli, P. Marchetti, S. Takasawa, H. Okamoto and E. Ferrannini (2001). "Anti-CD38 autoimmunity in patients with chronic autoimmune thyroiditis or Graves' disease." Clin Exp Immunol 126(3): 426-431. Antonelli, A. and E. Ferrannini (2004). "CD38 autoimmunity: recent advances and relevance to human diabetes." J Endocrinol Invest 27(7): 695-707. Banys-Paluchowski, M., I. Witzel, B. Aktas, P. A. Fasching, A. Hartkopf, W. Janni, S. Kasimir-Bauer, K. Pantel, G. Schon, B. Rack, S. Riethdorf, E. F. Solomayer, T. Fehm and V. Muller (2019). "The prognostic relevance of urokinase-type plasminogen activator (uPA) in the blood of patients with metastatic breast cancer." Sci Rep 9(1): 2318. Biopharmadive.com. (2021). Blacher, E., B. Ben Baruch, A. Levy, N. Geva, K. D. Green, S. Garneau-Tsodikova, M. Fridman and R. Stein (2015). "Inhibition of glioma progression by a newly discovered CD38 inhibitor." Int J Cancer 136(6): 1422-1433. Blacher, E., T. Dadali, A. Bespalko, V. J. Haupenthal, M. O. Grimm, T. Hartmann, F. E. Lund, R. Stein and A. Levy (2015). "Alzheimer's disease pathology is attenuated in a CD38-deficient mouse model." Ann Neurol 78(1): 88-103. Boini, K. M., M. Xia, J. Xiong, C. Li, L. P. Payne and P. L. Li (2012). "Implication of CD38 gene in podocyte epithelial-to-mesenchymal transition and glomerular sclerosis." J Cell Mol Med 16(8): 1674-1685. Boison, D. and G. G. Yegutkin (2019). "Adenosine Metabolism: Emerging Concepts for Cancer Therapy." Cancer Cell 36(6): 582-596. Bolt, S., E. Routledge, I. Lloyd, L. Chatenoud, H. Pope, S. D. Gorman, M. Clark and H. Waldmann (1993). "The generation of a humanized, non-mitogenic CD3 monoclonal antibody which retains in vitro immunosuppressive properties." Eur J Immunol 23(2): 403-411. Boslett, J., C. Hemann, F. L. Christofi and J. L. Zweier (2018). "Characterization of CD38 in the major cell types of the heart: endothelial cells highly express CD38 with activation by hypoxia-reoxygenation triggering NAD(P)H depletion." Am J Physiol Cell Physiol 314(3): C297-C309. Brischwein, K., B. Schlereth, B. Guller, C. Steiger, A. Wolf, R. Lutterbuese, S. Offner, M. Locher, T. Urbig, T. Raum, P. Kleindienst, P. Wimberger, R. Kimmig, I. Fichtner, P. Kufer, R. Hofmeister, A. J. da Silva and P. A. Baeuerle (2006). "MT110: a novel bispecific single-chain antibody construct with high efficacy in eradicating established tumors." Mol Immunol 43(8): 1129-1143. Bu, X., J. Kato, J. A. Hong, M. J. Merino, D. S. Schrump, F. E. Lund and J. Moss (2018). "CD38 knockout suppresses tumorigenesis in mice and clonogenic growth of human lung cancer cells." Carcinogenesis 39(2): 242-251. Burgler, S. (2015). "Role of CD38 Expression in Diagnosis and Pathogenesis of Chronic Lymphocytic Leukemia and Its Potential as Therapeutic Target." Crit Rev Immunol 35(5): 417-432. Burgler, S., A. Gimeno, A. Parente-Ribes, D. Wang, A. Os, S. Devereux, P. Jebsen, B. Bogen, G. E. Tjonnfjord and L. A. Munthe (2015). "Chronic lymphocytic leukemia cells express CD38 in response to Th1 cell-derived IFN-gamma by a T-bet-dependent mechanism." J Immunol 194(2): 827-835. Calabretta, E. and C. Carlo-Stella (2020). "The Many Facets of CD38 in Lymphoma: From Tumor-Microenvironment Cell Interactions to Acquired Resistance to Immunotherapy." Cells 9(4). Camacho-Pereira, J., M. G. Tarrago, C. C. S. Chini, V. Nin, C. Escande, G. M. Warner, A. S. Puranik, R. A. Schoon, J. M. Reid, A. Galina and E. N. Chini (2016). "CD38 Dictates Age-Related NAD Decline and Mitochondrial Dysfunction through an SIRT3-Dependent Mechanism." Cell Metab 23(6): 1127-1139. Cambronne, X. A. and W. L. Kraus (2020). "Location, Location, Location: Compartmentalization of NAD(+) Synthesis and Functions in Mammalian Cells." Trends Biochem Sci 45(10): 858-873. Chatenoud, L., C. Ferran, C. Legendre, I. Thouard, S. Merite, A. Reuter, Y. Gevaert, H. Kreis, P. Franchimont and J. F. Bach (1990). "In vivo cell activation following OKT3 administration. Systemic cytokine release and modulation by corticosteroids." Transplantation 49(4): 697-702. Chen, X., J. L. Zaro and W. C. Shen (2013). "Fusion protein linkers: property, design and functionality." Adv Drug Deliv Rev 65(10): 1357-1369. Chini, E. N., C. C. S. Chini, J. M. Espindola Netto, G. C. de Oliveira and W. van Schooten (2018). "The Pharmacology of CD38 / NADase: An Emerging Target in Cancer and Diseases of Aging." Trends Pharmacol Sci 39(4): 424-436. Chmielewski, J. P., S. C. Bowlby, F. B. Wheeler, L. Shi, G. Sui, A. L. Davis, T. D. Howard, R. B. D'Agostino, Jr., L. D. Miller, S. J. Sirintrapun, S. D. Cramer and S. J. Kridel (2018). "CD38 Inhibits Prostate Cancer Metabolism and Proliferation by Reducing Cellular NAD(+) Pools." Mol Cancer Res 16(11): 1687-1700. Chmielewski, M., A. Hombach, C. Heuser, G. P. Adams and H. Abken (2004). "T cell activation by antibody-like immunoreceptors: increase in affinity of the single-chain fragment domain above threshold does not increase T cell activation against antigen-positive target cells but decreases selectivity." J Immunol 173(12): 7647-7653. Chothia, C. and A. M. Lesk (1987). "Canonical structures for the hypervariable regions of immunoglobulins." J Mol Biol 196(4): 901-917. Chu, S. Y., I. Vostiar, S. Karki, G. L. Moore, G. A. Lazar, E. Pong, P. F. Joyce, D. E. Szymkowski and J. R. Desjarlais (2008). "Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcgammaRIIb with Fc-engineered antibodies." Mol Immunol 45(15): 3926-3933. Covarrubias, A. J., A. Kale, R. Perrone, J. A. Lopez-Dominguez, A. O. Pisco, H. G. Kasler, M. S. Schmidt, I. Heckenbach, R. Kwok, C. D. Wiley, H. S. Wong, E. Gibbs, S. S. Iyer, N. Basisty, Q. Wu, I. J. Kim, E. Silva, K. Vitangcol, K. O. Shin, Y. M. Lee, R. Riley, I. Ben-Sahra, M. Ott, B. Schilling, M. Scheibye-Knudsen, K. Ishihara, S. R. Quake, J. Newman, C. Brenner, J. Campisi and E. Verdin (2020). "Senescent cells promote tissue NAD(+) decline during ageing via the activation of CD38(+) macrophages." Nat Metab 2(11): 1265-1283. Dall'Acqua, W. F., P. A. Kiener and H. Wu (2006). "Properties of human IgG1s engineered for enhanced binding to the neonatal Fc receptor (FcRn)." J Biol Chem 281(33): 23514-23524. Damle , RN , T Wasil , F Fais , F Ghiotto , A Valetto , SL Allen , A Buchbinder , D Budman , K Dittmar , J Kolitz , SM Lichtman , P Schulman , VP Vinciguerra , KR Rai , M Ferrarini and N Chiorazzi (1999). "Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia." Blood 94(6): 1840–1847. Deaglio , S. , K. Mehta and F. Malavasi (2001). "Human CD38: a (r)evolutionary story of enzymes and receptors." Lung Res 25(1):1-12. Deshpande, DA, AGP Guedes, FE Lund, S Subramanian, TF Walseth and MS Kannan (2017). "CD38 in the pathogenesis of allergic airway disease: Potential therapeutic targets." Pharmacol Ther 172:116–126. Diebolder, CA, FJ Beurskens, RN de Jong, RI Koning, K. Strumane, MA Lindorfer, M. Voorhorst, D. Ugurlar, S. Rosati, AJ Heck, JG van de Winkel, IA Wilson, AJ Koster, RP Taylor, EO Saphire, DR Burton, J. Schuurman, PW Parren and PW (2014). "Complement is activated by IgG hexamers assembled at the cell surface." Science 343(6176): 1260-1263. Dimopoulos, MA, A. Oriol, H. Nahi, J. San Miguel, NJ Bahlis, SZ Usmani, N. Rabin, RZ Orlowski, M. Komarnicki, K. Suzuki, T. Plesner, SS Yoon, D. Ben Yehuda, PG Richardson, H. Goldschmidt, D. Reecekhar, S. Lihokhar, N. Z. K. K. K. O’Rourke, C. Chiu, X. Qin, M. Guckert, T. Ahmadi, P. Moreau and P. Investigators (2016). "Daratumumab, Lenalidomide, and Dexamethasone for Multiple Myeloma." N Engl J Med 375(14): 1319-1331. Drent, E., RW Groen, WA North, M. Themeli, JJ Lammerts van Bueren, PW Parren, J. Kuball, Z. Sebestyen, H. Yuan, J. de Bruijn, NW van de Donk, AC Martens, HM Lokhorst and T. Mutis (2016). "Pre-clinical evaluation of CD38 chimeric antigen receptor engineered T cells for the treatment of multiple myeloma." Haematologica 101(5): 616-625. Drent, E., M. Themeli, R. Poels, R. de Jong-Korlaar, H. Yuan, J. de Bruijn, ACM Martens, S. Zweegman, N. van de Donk, RWJ Groen, HM Lokhorst and T. Mutis (2017). "A Rational Strategy for Reducing On-Target Off-Tumor Effects of CD38-Chimeric Antigen Receptors by Affinity Optimization." Mol Ther 25(8): 1946-1958. Fayon, M., C. Martinez-Cingolani, A. Abecassis, N. Roders, E. Nelson, C. Choisy, A. Talbot, A. Bensussan, J. P. Fermand, B. Arnulf and J. C. Bories (2021). "Bi38-3 is a novel CD38 / CD3 bispecific T-cell engager with low toxicity for the treatment of multiple myeloma." Haematologica 106(4): 1193-1197. Frankel, S. R. and P. A. Baeuerle (2013). "Targeting T cells to tumor cells using bispecific antibodies." Curr Opin Chem Biol 17(3): 385-392. Fumey, W., J. Koenigsdorf, V. Kunick, S. Menzel, K. Schutze, M. Unger, L. Schriewer, F. Haag, G. Adam, A. Oberle, M. Binder, R. Fliegert, A. Guse, Y. J. Zhao, H. Cheung Lee, F. Malavasi, F. Goldbaum, R. van Hegelsom, C. Stortelers, P. Bannas and F. Koch-Nolte (2017). "Nanobodies effectively modulate the enzymatic activity of CD38 and allow specific imaging of CD38(+) tumors in mouse models in vivo." Sci Rep 7(1): 14289. Ge, Y., Y. Long, S. Xiao, L. Liang, Z. He, C. Yue, X. Wei and Y. Zhou (2019). "CD38 affects the biological behavior and energy metabolism of nasopharyngeal carcinoma cells." Int J Oncol 54(2): 585-599. Glaria, E. and A. F. Valledor (2020). "Roles of CD38 in the Immune Response to Infection." Cells 9(1). Guan, X. H., X. Hong, N. Zhao, X. H. Liu, Y. F. Xiao, T. T. Chen, L. B. Deng, X. L. Wang, J. B. Wang, G. J. Ji, M. Fu, K. Y. Deng and H. B. Xin (2017). "CD38 promotes angiotensin II-induced cardiac hypertrophy." J Cell Mol Med 21(8): 1492-1502. Guedes, A. G., M. Dileepan, J. A. Jude, D. A. Deshpande, T. F. Walseth and M. S. Kannan (2020). "Role of CD38 / cADPR signaling in obstructive pulmonary diseases." Curr Opin Pharmacol 51: 29-33. Guerreiro, S., A. L. Privat, L. Bressac and D. Toulorge (2020). "CD38 in Neurodegeneration and Neuroinflammation." Cells 9(2). Gunasekaran, K., M. Pentony, M. Shen, L. Garrett, C. Forte, A. Woodward, S. B. Ng, T. Born, M. Retter, K. Manchulenko, H. Sweet, I. N. Foltz, M. Wittekind and W. Yan (2010). "Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG." J Biol Chem 285(25): 19637-19646. Henriques, A., I. Silva, L. Ines, M. M. Souto-Carneiro, M. L. Pais, H. Trindade, J. A. da Silva and A. Paiva (2016). "CD38, CD81 and BAFFR combined expression by transitional B cells distinguishes active from inactive systemic lupus erythematosus." Clin Exp Med 16(2): 227-232. Higashida, H. and T. Munesue (2013). "[CD38 and autism spectrum disorders]." No To Hattatsu 45(6): 431-435. Hinton, P. R., J. M. Xiong, M. G. Johlfs, M. T. Tang, S. Keller and N. Tsurushita (2006). "An engineered human IgG1 antibody with longer serum half-life." J Immunol 176(1): 346-356. Hipp, S., Y. T. Tai, D. Blanset, P. Deegen, J. Wahl, O. Thomas, B. Rattel, P. J. Adam, K. C. Anderson and M. Friedrich (2017). "A novel BCMA / CD3 bispecific T-cell engager for the treatment of multiple myeloma induces selective lysis in vitro and in vivo." Leukemia 31(8): 1743-1751. Hogan, K. A., C. C. S. Chini and E. N. Chini (2019). "The Multi-faceted Ecto-enzyme CD38: Roles in Immunomodulation, Cancer, Aging, and Metabolic Diseases." Front Immunol 10: 1187. Jiang, Z., D. Wu, S. Lin and P. Li (2016). "CD34 and CD38 are prognostic biomarkers for acute B lymphoblastic leukemia." Biomark Res 4: 23. Jiao, Y., M. Yi, L. Xu, Q. Chu, Y. Yan, S. Luo and K. Wu (2020). "CD38: targeted therapy in multiple myeloma and therapeutic potential for solid cancers." Expert Opin Investig Drugs 29(11): 1295-1308. Joosse, M. E., C. L. Menckeberg, L. F. de Ruiter, H. R. C. Raatgeep, L. A. van Berkel, Y. Simons-Oosterhuis, I. Tindemans, A. F. M. Muskens, R. W. Hendriks, R. M. Hoogenboezem, T. Cupedo, L. de Ridder, J. C. Escher, S. Veenbergen and J. N. Samsom (2019). "Frequencies of circulating regulatory TIGIT(+)CD38(+) effector T cells correlate with the course of inflammatory bowel disease." Mucosal Immunol 12(1): 154-163. Kang, B. E., J. Y. Choi, S. Stein and D. Ryu (2020). "Implications of NAD(+) boosters in translational medicine." Eur J Clin Invest 50(10): e13334. Kar, A., S. Mehrotra and S. Chatterjee (2020). "CD38: T Cell Immuno-Metabolic Modulator." Cells 9(7). Karimi-Busheri, F., V. Zadorozhny, T. Li, H. Lin, D. L. Shawler and H. Fakhrai (2011). "Pivotal role of CD38 biomarker in combination with CD24, EpCAM, and ALDH for identification of H460 derived lung cancer stem cells." J Stem Cells 6(1): 9-20. Kim, B. J., Y. M. Choi, S. Y. Rah, D. R. Park, S. A. Park, Y. J. Chung, S. M. Park, J. K. Park, K. Y. Jang and U. H. Kim (2015). "Seminal CD38 is a pivotal regulator for fetomaternal tolerance." Proc Natl Acad Sci U S A 112(5): 1559-1564. Kim, B. J., D. R. Park, T. S. Nam, S. H. Lee and U. H. Kim (2015). "Seminal CD38 Enhances Human Sperm Capacitation through Its Interaction with CD31." PLoS One 10(9): e0139110. Kinder, M., A. R. Greenplate, K. D. Grugan, K. L. Soring, K. A. Heeringa, S. G. McCarthy, G. Bannish, M. Perpetua, F. Lynch, R. E. Jordan, W. R. Strohl and R. J. Brezski (2013). "Engineered protease-resistant antibodies with selectable cell-killing functions." J Biol Chem 288(43): 30843-30854. Klein, C., C. Sustmann, M. Thomas, K. Stubenrauch, R. Croasdale, J. Schanzer, U. Brinkmann, H. Kettenberger, J. T. Regula and W. Schaefer (2012). "Progress in overcoming the chain association issue in bispecific heterodimeric IgG antibodies." MAbs 4(6): 653-663. Labrijn, A. F., J. I. Meesters, B. E. de Goeij, E. T. van den Bremer, J. Neijssen, M. D. van Kampen, K. Strumane, S. Verploegen, A. Kundu, M. J. Gramer, P. H. van Berkel, J. G. van de Winkel, J. Schuurman and P. W. Parren (2014). "Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange." Proc Natl Acad Sci U S A 110(13): 5145-5150. Lam, J. H., H. H. M. Ng, C. J. Lim, X. N. Sim, F. Malavasi, H. Li, J. J. H. Loh, K. Sabai, J. K. Kim, C. C. H. Ong, T. Loh, W. Q. Leow, S. P. Choo, H. C. Toh, S. Y. Lee, C. Y. Chan, V. Chew, T. S. Lim, J. Yeong and T. K. H. Lim (2019). "Expression of CD38 on Macrophages Predicts Improved Prognosis in Hepatocellular Carcinoma." Front Immunol 10: 2093. Lefranc, M. P., C. Pommie, M. Ruiz, V. Giudicelli, E. Foulquier, L. Truong, V. Thouvenin-Contet and G. Lefranc (2003). "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains." Dev Comp Immunol 27(1): 55-77. Li, J., N. J. Stagg, J. Johnston, M. J. Harris, S. A. Menzies, D. DiCara, V. Clark, M. Hristopoulos, R. Cook, D. Slaga, R. Nakamura, L. McCarty, S. Sukumaran, E. Luis, Z. Ye, T. D. Wu, T. Sumiyoshi, D. Danilenko, G. Y. Lee, K. Totpal, D. Ellerman, I. Hotzel, J. R. James and T. T. Junttila (2017). "Membrane-Proximal Epitope Facilitates Efficient T Cell Synapse Formation by Anti-FcRH5 / CD3 and Is a Requirement for Myeloma Cell Killing." Cancer Cell 31(3): 383-395. Liao, S., S. Xiao, H. Chen, M. Zhang, Z. Chen, Y. Long, L. Gao, G. Zhu, J. He, S. Peng, W. Xiong, Z. Zeng, Z. Li, M. Zhou, X. Li, J. Ma, M. Wu, J. Xiang, G. Li and Y. Zhou (2017). "CD38 enhances the proliferation and inhibits the apoptosis of cervical cancer cells by affecting the mitochondria functions." Mol Carcinog 56(10): 2245-2257. Liao, S., S. Xiao, G. Zhu, D. Zheng, J. He, Z. Pei, G. Li and Y. Zhou (2014). "CD38 is highly expressed and affects the PI3K / Akt signaling pathway in cervical cancer." Oncol Rep 32(6): 2703-2709. Lin, W. W., Y. C. Lu, C. H. Chuang and T. L. Cheng (2020). "Ab locks for improving the selectivity and safety of antibody drugs." J Biomed Sci 27(1): 76. Lins, L., E. Farias, C. Brites-Alves, A. Torres, E. M. Netto and C. Brites (2017). "Increased expression of CD38 and HLADR in HIV-infected patients with oral lesion." J Med Virol 89(10): 1782-1787. Liu, X., T. R. Grogan, H. Hieronymus, T. Hashimoto, J. Mottahedeh, D. Cheng, L. Zhang, K. Huang, T. Stoyanova, J. W. Park, R. O. Shkhyan, B. Nowroozizadeh, M. B. Rettig, C. L. Sawyers, D. Elashoff, S. Horvath, J. Huang, O. N. Witte and A. S. Goldstein (2016). "Low CD38 Identifies Progenitor-like Inflammation-Associated Luminal Cells that Can Initiate Human Prostate Cancer and Predict Poor Outcome." Cell Rep 17(10): 2596-2606. Lopatina, O., A. Inzhutova, A. B. Salmina and H. Higashida (2012). "The roles of oxytocin and CD38 in social or parental behaviors." Front Neurosci 6: 182. Ma, S. R., W. W. Deng, J. F. Liu, L. Mao, G. T. Yu, L. L. Bu, A. B. Kulkarni, W. F. Zhang and Z. J. Sun (2017). "Blockade of adenosine A2A receptor enhances CD8(+) T cells response and decreases regulatory T cells in head and neck squamous cell carcinoma." Mol Cancer 16(1): 99. Malavasi , F. , S. Deaglio , R. Damle , G. Cutrona , M. Ferrarini and N. Chiorazzi (2011). "CD38 and chronic lymphocytic leukemia: a decade later." Blood 118(13):3470–3478. Malavasi , F. , S. Deaglio , A. Funaro , E. Ferrero , AL Horenstein , E. Ortolan , T. Vaisitti and S. Aydin (2008). "Evolution and function of the ADP ribosyl cyclase / CD38 gene family in physiology and pathology." Physiol Rev 88(3):841–886. Malavasi , F. , A. Funaro , S. Roggero , A. Horenstein , L. Calosso and K. Mehta (1994). "Human CD38: a glycoprotein in search of a function." Immunology Today 15(3): 95-97. Mallone , R. , E. Ortolan , S. Pinach , M. Volante , MM Zanone , G. Bruno , G. Baj , T. Lohmann , P. Cavallo-Perin and F. Malavasi (2002). "Anti-CD38 autoantibodies: characterization in new-onset type I diabetes and latent autoimmune diabetes of the adult (LADA) and comparison with other islet autoantibodies." Diabetologia 45(12): 1667-1677. Mallone, R. and P. C. Perin (2006). "Anti-CD38 autoantibodies in type? diabetes." Diabetes Metab Res Rev 22(4): 284-294. Marlein, C. R., R. E. Piddock, J. J. Mistry, L. Zaitseva, C. Hellmich, R. H. Horton, Z. Zhou, M. J. Auger, K. M. Bowles and S. A. Rushworth (2019). "CD38-Driven Mitochondrial Trafficking Promotes Bioenergetic Plasticity in Multiple Myeloma." Cancer Res 79(9): 2285-2297. May, C., P. Sapra and H. P. Gerber (2012). "Advances in bispecific biotherapeutics for the treatment of cancer." Biochem Pharmacol 84(9): 1105-1112. Mehta, K., U. Shahid and F. Malavasi (1996). "Human CD38, a cell-surface protein with multiple functions." FASEB J 10(12): 1408-1417. Moore, G. L., C. Bautista, E. Pong, D. H. Nguyen, J. Jacinto, A. Eivazi, U. S. Muchhal, S. Karki, S. Y. Chu and G. A. Lazar (2011). "A novel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens." MAbs 3(6): 546-557. Morandi, F., I. Airoldi, D. Marimpietri, C. Bracci, A. C. Faini and R. Gramignoli (2019). "CD38, a Receptor with Multifunctional Activities: From Modulatory Functions on Regulatory Cell Subsets and Extracellular Vesicles, to a Target for Therapeutic Strategies." Cells 8(12). Munoz, P., M. Mittelbrunn, H. de la Fuente, M. Perez-Martinez, A. Garcia-Perez, A. Ariza-Veguillas, F. Malavasi, M. Zubiaur, F. Sanchez-Madrid and J. Sancho (2008). "Antigen-induced clustering of surface CD38 and recruitment of intracellular CD38 to the immunologic synapse." Blood 111(7): 3653-3664. Nooka, A. K., J. L. Kaufman, C. C. Hofmeister, N. S. Joseph, T. L. Heffner, V. A. Gupta, H. C. Sullivan, A. S. Neish, M. V. Dhodapkar and S. Lonial (2019). "Daratumumab in multiple myeloma." Cancer 125(14): 2364-2382. Orciani, M., O. Trubiani, S. Guarnieri, E. Ferrero and R. Di Primio (2008). "CD38 is constitutively expressed in the nucleus of human hematopoietic cells." J Cell Biochem 105(3): 905-912. Palumbo, A., A. Chanan-Khan, K. Weisel, AK Nooka, T. Masszi, M. Beksac, I. Spicka, V. Hungria, M. Munder, MV Mateos, TM Mark, M. Qi, J. Schecter, H. Amin, X. Qin, W. Deraedt, T. Ahmadi, A. P. Spencerd, and C. Sonneveld. Investigators (2016). "Daratumumab, Bortezomib, and Dexamethasone for Multiple Myeloma." N Engl J Med 375(8): 754-766. Perenkov, AD, DV Novikov, NA Sakharnov, AV Aliasova, OV Utkin, A. Baryshnikov and VV Novikov (2012). "[Heterogeneous expression of CD38 gene in tumor tissue in patients with colorectal cancer]." Mol Biol (Moscow) 46(5): 786-791. Petkova, S. B., S. Akilesh, T. J. Sproule, G. J. Christianson, H. Al Khabbaz, A. C. Brown, L. G. Presta, Y. G. Meng and D. C. Roopenian (2006). "Enhanced half-life of genetically engineered human IgG1 antibodies in a humanized FcRn mouse model: potential application in humorally mediated autoimmune disease." Int Immunol 18(12): 1759-1769. Quarona, V., G. Zaccarello, A. Chillemi, E. Brunetti, V. K. Singh, E. Ferrero, A. Funaro, A. L. Horenstein and F. Malavasi (2013). "CD38 and CD157: a long journey from activation markers to multifunctional molecules." Cytometry B Clin Cytom 84(4): 207-217. Ridgway, J. B., L. G. Presta and P. Carter (1996). "'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization." Protein Eng 9(7): 617-621. Salmina, A. B., O. Lopatina, N. V. Kuvacheva and H. Higashida (2013). "Integrative neurochemistry and neurobiology of social recognition and behavior analyzed with respect to CD38-dependent brain oxytocin secretion." Curr Top Med Chem 13(23): 2965-2977. Saltarella, I., V. Desantis, A. Melaccio, A. G. Solimando, A. Lamanuzzi, R. Ria, C. T. Storlazzi, M. A. Mariggio, A. Vacca and M. A. Frassanito (2020). "Mechanisms of Resistance to Anti-CD38 Daratumumab in Multiple Myeloma." Cells 9(1). Sanchez, E., M. Li, A. Kitto, J. Li, C. S. Wang, D. T. Kirk, O. Yellin, C. M. Nichols, M. P. Dreyer, C. P. Ahles, A. Robinson, E. Madden, G. N. Waterman, R. A. Swift, B. Bonavida, R. Boccia, R. A. Vescio, J. Crowley, H. Chen and J. R. Berenson (2012). "Serum B-cell maturation antigen is elevated in multiple myeloma and correlates with disease status and survival." Br J Haematol 158(6): 727-738. Scheraga, H. A. (2008). "From helix-coil transitions to protein folding." Biopolymers 89(5): 479-485. Schiavoni, I., C. Scagnolari, A. L. Horenstein, P. Leone, A. Pierangeli, F. Malavasi, C. M. Ausiello and G. Fedele (2018). "CD38 modulates respiratory syncytial virus-driven proinflammatory processes in human monocyte-derived dendritic cells." Immunology 154(1): 122-131. Schultz, M. B. and D. A. Sinclair (2016). "Why NAD(+) Declines during Aging: It's Destroyed." Cell Metab 23(6): 965-966. Schutze, K., K. Petry, J. Hambach, N. Schuster, W. Fumey, L. Schriewer, J. Rockendorf, S. Menzel, B. Albrecht, F. Haag, C. Stortelers, P. Bannas and F. Koch-Nolte (2018). "CD38-Specific Biparatopic Heavy Chain Antibodies Display Potent Complement-Dependent Cytotoxicity Against Multiple Myeloma Cells." Front Immunol 9: 2553. Seckinger , A , Delgado JA , Moser S , Moreno L , Neuber B , Grab A , Lipp , Merino J , Prosper F , Emde M , Delon C , Latzko M , Gianotti R , Luoend R , Murr R , Hosse RJ , Harnisch LJ , Bacac M , Fauti T , Klein C , Zabaleta A , Hillengass J, Cavalcanti-Adam EA, Ho AD, Hundemer M, San Miguel JF, Strein K, Umana P, Hose D, Paiva B and Vu MD (2017). "Target Expression, Generation, Preclinical Activity, and Pharmacokinetics of the BCMA-T Cell Bispecific Antibody EM801 for Multiple Myeloma Treatment." Cancer Cell 31(3): 396-410. Shields , RL , AK Namenuk , K Hong , YG Meng , J Rae , J Briggs , D Xie , J Lai , A Stadlen , B Li , JA Fox and LG Presta ( 2001 ). "High resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1 variants with improved binding to the Fc gamma R." J Biol Chem 276(9):6591–6604. Shields, R. L., A. K. Namenuk, K. Hong, Y. G. Meng, J. Rae, J. Briggs, D. Xie, J. Lai, A. Stadlen, B. Li, J. A. Fox and L. G. Presta (2001). "High resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1 variants with improved binding to the Fc gamma R." J Biol Chem 276(9): 6591-6604. Stone, L. (2017). "Prostate cancer: On the down-low - low luminal cell CD38 expression is prognostic." Nat Rev Urol 14(3): 133. Su, A. I., T. Wiltshire, S. Batalov, H. Lapp, K. A. Ching, D. Block, J. Zhang, R. Soden, M. Hayakawa, G. Kreiman, M. P. Cooke, J. R. Walker and J. B. Hogenesch (2004). "A gene atlas of the mouse and human protein-encoding transcriptomes." Proc Natl Acad Sci U S A 101(16): 6062-6067. Tam, S. H., S. G. McCarthy, A. A. Armstrong, S. Somani, S. J. Wu, X. Liu, A. Gervais, R. Ernst, D. Saro, R. Decker, J. Luo, G. L. Gilliland, M. L. Chiu and B. J. Scallon (2017). "Functional, Biophysical, and Structural Characterization of Human IgG1 and IgG4 Fc Variants with Ablated Immune Functionality." Antibodies (Basel) 6(3). Usmani, S. Z., B. M. Weiss, T. Plesner, N. J. Bahlis, A. Belch, S. Lonial, H. M. Lokhorst, P. M. Voorhees, P. G. Richardson, A. Chari, A. K. Sasser, A. Axel, H. Feng, C. M. Uhlar, J. Wang, I. Khan, T. Ahmadi and H. Nahi (2016). "Clinical efficacy of daratumumab monotherapy in patients with heavily pretreated relapsed or refractory multiple myeloma." Blood 128(1): 37-44. Vafa, O., G. L. Gilliland, R. J. Brezski, B. Strake, T. Wilkinson, E. R. Lacy, B. Scallon, A. Teplyakov, T. J. Malia and W. R. Strohl (2014). "An engineered Fc variant of an IgG eliminates all immune effector functions via structural perturbations." Methods 65(1): 114-126. van de Donk, N. and S. Z. Usmani (2018). "CD38 Antibodies in Multiple Myeloma: Mechanisms of Action and Modes of Resistance." Front Immunol 9: 2134. Wang, H., G. Kaur, A. I. Sankin, F. Chen, F. Guan and X. Zang (2019). "Immune checkpoint blockade and CAR-T cell therapy in hematologic malignancies." J Hematol Oncol 12(1): 59. Wang, H., S. Li, G. Zhang, H. Wu and X. Chang (2019). "Potential therapeutic effects of cyanidin-3-O-glucoside on rheumatoid arthritis by relieving inhibition of CD38+ NK cells on Treg cell differentiation." Arthritis Res Ther 21(1): 220. Wang, L., S. S. Hoseini, H. Xu, V. Ponomarev and N. K. Cheung (2019). "Silencing Fc Domains in T cell-Engaging Bispecific Antibodies Improves T-cell Trafficking and Antitumor Potency." Cancer Immunol Res 7(12): 2013-2024. Wikipedia. (2021). "Catumaxomab." Worn, A. and A. Pluckthun (2001). "Stability engineering of antibody single-chain Fv fragments." J Mol Biol 305(5): 989-1010. Wu, C., X. Jin, G. Tsueng, C. Afrasiabi and A. I. Su (2016). "BioGPS: building your own mash-up of gene annotations and expression profiles." Nucleic Acids Res 44(D1): D313-316. Wu, C., L. Zhang, Q. R. Brockman, F. Zhan and L. Chen (2019). "Chimeric antigen receptor T cell therapies for multiple myeloma." J Hematol Oncol 12(1): 120. Wu, T. T. and E. A. Kabat (1970). "An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity." J Exp Med 132(2): 211-250. Wursch, D., C. E. Ormsby, D. P. Romero-Rodriguez, G. Olvera-Garcia, J. Zuniga, W. Jiang, S. Perez-Patrigeon and E. Espinosa (2016). "CD38 Expression in a Subset of Memory T Cells Is Independent of Cell Cycling as a Correlate of HIV Disease Progression." Dis Markers 2016: 9510756. Xu, D., M. L. Alegre, S. S. Varga, A. L. Rothermel, A. M. Collins, V. L. Pulito, L. S. Hanna, K. P. Dolan, P. W. Parren, J. A. Bluestone, L. K. Jolliffe and R. A. Zivin (2000). "In vitro characterization of five humanized OKT3 effector function variant antibodies." Cell Immunol 200(1): 16-26. Xu, L., D. Chen, C. Lu, X. Liu, G. Wu and Y. Zhang (2015). "Advanced Lung Cancer Is Associated with Decreased Expression of Perforin, CD95, CD38 by Circulating CD3+CD8+ T Lymphocytes." Ann Clin Lab Sci 45(5): 528-532. Yarbro, J. R., R. S. Emmons and B. D. Pence (2020). "Macrophage Immunometabolism and Inflammaging: Roles of Mitochondrial Dysfunction, Cellular Senescence, CD38, and NAD." Immunometabolism 2(3): e200026. Yeong, J., J. C. T. Lim, B. Lee, H. Li, N. Chia, C. C. H. Ong, W. K. Lye, T. C. Putti, R. Dent, E. Lim, A. A. Thike, P. H. Tan and J. Iqbal (2018). "High Densities of Tumor-Associated Plasma Cells Predict Improved Prognosis in Triple Negative Breast Cancer." Front Immunol 9: 1209. Yu, S., M. Yi, S. Qin and K. Wu (2019). "Next generation chimeric antigen receptor T cells: safety strategies to overcome toxicity." Mol Cancer 18(1): 125. Zalevsky, J., A. K. Chamberlain, H. M. Horton, S. Karki, I. W. Leung, T. J. Sproule, G. A. Lazar, D. C. Roopenian and J. R. Desjarlais (2010). "Enhanced antibody half-life improves in vivo activity." Nat Biotechnol 28(2): 157-159. Zambello, R., G. Barila, S. Manni, F. Piazza and G. Semenzato (2020). "NK cells and CD38: Implication for (Immuno)Therapy in Plasma Cell Dyscrasias." Cells 9(3). Zeijlemaker, W., T. Grob, R. Meijer, D. Hanekamp, ​​A. Kelder, JC Carbaat-Ham, YJM Oussoren-Brockhoff, AN Snel, D. Veldhuizen, WJ Scholten, J. Maertens, DA Breems, T. Pabst, MG Manz, Jder Velden, JJ, van Slompden, F. Slompden. Preijers, J. Cloos, AA van de Loosdrecht, B. Lowenberg, PJM Valk, M. Jongen-Lavrencic, GJ Ossenkoppele and GJ Schuurhuis (2019). "CD34(+)CD38(-) leukemic stem cell frequency to predict outcome in acute myeloid leukemia." Leukemia 33(5): 1102-1112. Zhang, D., AA Armstrong, SH Tam, SG McCarthy, J. Luo, GL Gilliland and ML Chiu (2017). "Functional optimization of agonistic antibodies to OX40 receptor with novel Fc mutations to promote antibody multimerization." MAbs 9: 1129-1142. Zhang, M., J. Yang, J. Zhou, W. Gao, Y. Zhang, Y. Lin, H. Wang, Z. Ruan and B. Ni (2019). "Prognostic Values of CD38(+)CD101(+)PD1(+)CD8(+) T Cells in Pancreatic Cancer." Immunol Invest 48(5): 466-479. Zheng, D., S. Liao, G. Zhu, G. Luo, S. Xiao, J. He, Z. Pei, G. Li and Y. Zhou (2016). "CD38 is a putative functional marker for side population cells in human nasopharyngeal carcinoma cell lines." Mol Carcinog 55(3): 300-311. Zuch de Zafra, C. L., F. Fajardo, W. Zhong, M. J. Bernett, U. S. Muchhal, G. L. Moore, J. Stevens, R. Case, J. T. Pearson, S. Liu, P. L. McElroy, J. Canon, J. R. Desjarlais, A. Coxon, M. Balazs and O. Nolan-Stevaux (2019). "Targeting Multiple Myeloma with AMG 424, a Novel Anti-CD38 / CD3 Bispecific T-cell-recruiting Antibody Optimized for Cytotoxicity and Cytokine Release." Clin Cancer Res 25(13): 3921-3933.

Claims

1. below: a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are selected from the following, respectively: SEQ ID NOs: 64, 65, and 66; SEQ ID NOs: 64, 83, and 84; SEQ ID NOs: 64, 65, and 85; and SEQ ID NOs: 64, 65, and 86; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 are selected from the following, respectively: SEQ ID NOs: 61, 62, and 63; SEQ ID NOs: 76, 77, and 78; SEQ ID NOs: 79, 80, and 81; and SEQ ID NOs: 82, 62, and 78; An anti-CD38 antibody or an antigen-binding fragment thereof comprising:

2. 2. The anti-CD38 antibody or antigen-binding fragment of claim 1, comprising a heavy chain sequence comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 12-16, or an antigen-binding fragment thereof, and a light chain sequence comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 7-11, or an antigen-binding fragment thereof.

3. An anti-CD38 antibody or antigen-binding fragment thereof comprising light chain sequences and heavy chain sequences comprising the following, respectively: SEQ ID NOs: 2 and 3; SEQ ID NOs: 7 and 12; SEQ ID NOs: 7 and 13; SEQ ID NOs: 7 and 14; SEQ ID NOs: 7 and 15; SEQ ID NOs: 7 and 16; SEQ ID NOs: 8 and 12; SEQ ID NOs: 8 and 13; SEQ ID NOs: 8 and 14; SEQ ID NOs: 8 and 15; SEQ ID NOs: 8 and 16; SEQ ID NOs: 9 and 12; SEQ ID NOs: 9 and 13; SEQ ID NOs: 9 and 14; SEQ ID NOs: 9 and 15; SEQ ID NOs: 9 and 16; SEQ ID NOs: 10 and 12; SEQ ID NOs: 10 and 13; SEQ ID NOs: 10 and 14; SEQ ID NOs: 10 and 15; SEQ ID NOs: 10 and 16; SEQ ID NOs: 11 and 12; SEQ ID NOs: 11 and 13; SEQ ID NOs: 11 and 14; SEQ ID NOs: 11 and 15; or SEQ ID NOs: 11 and 16.

4. An anti-CD38 antibody or antigen-binding fragment thereof comprising at least one variable heavy chain-only single domain or antigen-binding fragment thereof, wherein the at least one variable heavy chain-only single domain comprises HCDR1, HCDR2, and HCDR3 selected from the following, respectively: SEQ ID NOs: 67, 68, and 69; SEQ ID NOs: 67, 87, and 69; SEQ ID NOs: 67, 88, and 69; SEQ ID NOs: 67, 89, and 69; SEQ ID NOs: 67, 68, and 90; SEQ ID NOs: 70, 91, and 72; SEQ ID NOs: 70, 92, and 72; SEQ ID NOs: 70, 93, and 72; SEQ ID NOs: 94, 95, and 96; SEQ ID NOs: 70, 71, and 97; SEQ ID NOs: 73, 98, and 75; SEQ ID NOs: 73, 99, and 75; SEQ ID NOs: 73, 100, and 75; SEQ ID NOs: 73, 101, and 102; and SEQ ID NOs: 73, 103, and 104.

5. 5. The anti-CD38 antibody or antigen-binding fragment of claim 4, comprising at least one variable heavy chain-only single domain or antigen-binding fragment thereof, wherein the at least one variable heavy chain-only single domain comprises an amino acid sequence or antigen-binding fragment thereof having at least 85% identity to any one of SEQ ID NOs: 4 and 17-30.

6. below: a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are selected from the following, respectively: SEQ ID NOs: 105, 108, and 107; SEQ ID NOs: 105, 109, and 107; SEQ ID NOs: 105, 110, and 107; and SEQ ID NOs: 118, 119, and 120; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein said LCDR1, LCDR2, and LCDR3 are selected from the following: SEQ ID NOs: 114, 112, and 113; and SEQ ID NOs: 115, 116, and 117, respectively; An anti-CD3 antibody or an antigen-binding fragment thereof comprising:

7. 7. The anti-CD3 antibody or antigen-binding fragment of claim 6, comprising a heavy chain sequence comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 32-34 and 39, or an antigen-binding fragment thereof, and a light chain sequence comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 36-38, or an antigen-binding fragment thereof.

8. An anti-CD3 antibody or antigen-binding fragment thereof comprising light chain and heavy chain sequences comprising the following, respectively: SEQ ID NOs: 39 and 38; SEQ ID NOs: 41 and 40; SEQ ID NOs: 31 and 35; SEQ ID NOs: 31 and 36; SEQ ID NOs: 31 and 37; SEQ ID NOs: 32 and 35; SEQ ID NOs: 32 and 36; SEQ ID NOs: 32 and 37; SEQ ID NOs: 33 and 35; SEQ ID NOs: 33 and 36; SEQ ID NOs: 33 and 37; SEQ ID NOs: 34 and 35; SEQ ID NOs: 34 and 36; or SEQ ID NOs: 34 and 37.

9. below: (1) a first binding arm comprising: a first heavy chain fusion protein comprising, from N-terminus to C-terminus, an optional shield A, an optional protease sequence A, and an IgG heavy chain or antigen-binding fragment thereof; and a first light chain fusion protein comprising, from N-terminus to C-terminus, an optional shield B, an optional protease sequence B, and an IgG light chain or antigen-binding fragment thereof; including, where: the IgG heavy chain or antigen-binding fragment thereof of the first binding arm comprises an HCDR1, an HCDR2, and an HCDR3 selected from the following, respectively: SEQ ID NOs: 105, 108, and 107; SEQ ID NOs: 105, 109, and 107; SEQ ID NOs: 105, 110, and 107; and SEQ ID NOs: 118, 119, and 120; and the IgG light chain or antigen-binding fragment thereof of the first binding arm comprises an LCDR1, an LCDR2, and an LCDR3 selected from the following: SEQ ID NOs: 114, 112, and 113; and SEQ ID NOs: 115, 116, and 117, respectively; and (2) a second binding arm comprising: a second heavy chain fusion protein comprising, from N-terminus to C-terminus, an optional shield C, an optional protease sequence C, and an IgG heavy chain or antigen-binding fragment thereof; and a second light chain fusion protein comprising, from N-terminus to C-terminus, an optional shield D, an optional protease sequence D, and an IgG light chain or antigen-binding fragment thereof; including, where: the IgG heavy chain or antigen-binding fragment thereof of the second binding arm comprises an HCDR1, an HCDR2, and an HCDR3 selected from the following, respectively: SEQ ID NOs: 64, 65, and 66; SEQ ID NOs: 64, 83, and 84; SEQ ID NOs: 64, 65, and 85; and SEQ ID NOs: 64, 65, and 86; and The IgG light chain or antigen-binding fragment thereof of the second binding arm is, respectively: comprising LCDR1, LCDR2, and LCDR3 selected from SEQ ID NOs: 61, 62, and 63; SEQ ID NOs: 76, 77, and 78; SEQ ID NOs: 79, 80, and 81; and SEQ ID NOs: 82, 62, and 78, and wherein An anti-CD3 and anti-CD38 bispecific antibody, wherein the shields A to D are the same as or different from each other, and the protease sequences A to D are the same as or different from each other.

10. the IgG heavy chain or antigen-binding fragment thereof of the first binding arm comprises an amino acid sequence or antigen-binding fragment thereof having at least 85% identity to any one of SEQ ID NOs: 32-34 and 39; and the IgG light chain or antigen-binding fragment thereof of the first binding arm comprises a light chain sequence comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 36-38, or an antigen-binding fragment thereof; and the IgG heavy chain or antigen-binding fragment thereof of the second binding arm comprises a heavy chain sequence comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 12-16, or an antigen-binding fragment thereof; and 10. The anti-CD3 and anti-CD38 bispecific antibody of claim 9, wherein the IgG light chain, or antigen-binding fragment thereof, of the second binding arm comprises a light chain sequence comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 7 to 11, or an antigen-binding fragment thereof.

11. below: (1) a first binding arm comprising: a first heavy chain fusion protein comprising, from N-terminus to C-terminus, an optional shield A, an optional protease sequence A, and an IgG heavy chain or antigen-binding fragment thereof; and a first light chain fusion protein comprising, from N-terminus to C-terminus, an optional shield B, an optional protease sequence B, and an IgG light chain or antigen-binding fragment thereof; including, where: the IgG heavy chain or antigen-binding fragment thereof of the first binding arm comprises an HCDR1, an HCDR2, and an HCDR3 selected from the following, respectively: SEQ ID NOs: 105, 108, and 107; SEQ ID NOs: 105, 109, and 107; SEQ ID NOs: 105, 110, and 107; and SEQ ID NOs: 118, 119, and 120; and the IgG light chain or antigen-binding fragment thereof of the first binding arm comprises an LCDR1, an LCDR2, and an LCDR3 selected from the following: SEQ ID NOs: 114, 112, and 113; and SEQ ID NOs: 115, 116, and 117, respectively; and (2) a second binding arm comprising: a second heavy chain fusion protein comprising, from N-terminus to C-terminus, an optional shield C, an optional protease sequence C, and an IgG heavy chain (comprising at least one single domain of the variable heavy chain only or an antigen-binding fragment thereof); including, wherein the at least one variable heavy chain-only single domain or antigen-binding fragment thereof comprises an HCDR1, HCDR2, or HCDR3 selected from the following, respectively: SEQ ID NOs: 67, 68, and 69; SEQ ID NOs: 67, 87, and 69; SEQ ID NOs: 67, 88, and 69; SEQ ID NOs: 67, 89, and 69; SEQ ID NOs: 67, 68, and 90; SEQ ID NOs: 70, 91, and 72; SEQ ID NOs: 70, 92, and 72; SEQ ID NOs: 70, 93, and 72; SEQ ID NOs: 94, 95, and 96; SEQ ID NOs: 70, 71, and 97; SEQ ID NOs: 73, 98, and 75; SEQ ID NOs: 73, 99, and 75; SEQ ID NOs: 73, 100, and 75; SEQ ID NOs: 73, 101, and 102; and SEQ ID NOs: 73, 103, and 104; and an anti-CD3 and anti-CD38 bispecific antibody, wherein the shields A to C are the same as or different from each other, and the protease sequences A to C are the same as or different from each other.

12. the IgG heavy chain or antigen-binding fragment thereof of the first binding arm comprises an amino acid sequence or antigen-binding fragment thereof having at least 85% identity to any one of SEQ ID NOs: 32-34 and 39; the IgG light chain or antigen-binding fragment thereof of the first binding arm comprises a light chain sequence comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 36-38, or an antigen-binding fragment thereof; and 12. The anti-CD3 and anti-CD38 bispecific antibody of claim 11, wherein the at least one variable heavy chain-only single domain or antigen-binding fragment thereof comprises an amino acid sequence or antigen-binding fragment thereof having at least 85% identity to any one of SEQ ID NOs: 4 and 17 to 30.

13. 12. The bispecific antibody of claim 11 , wherein the first binding arm is monovalent and the second binding arm is monovalent, bivalent, or multivalent.

14. 12. The bispecific antibody of claim 11 , wherein the second binding arm comprises, in tandem, two or three IgG variable heavy chain-only single domains, optionally connected via one or more linker sequences.

15. 10. The bispecific antibody of claim 9, wherein shield A, shield B, shield C, and shield D are each independently selected from the amino acid sequences set forth in SEQ ID NOs: 42 to 52.

16. The bispecific antibody of claim 9, wherein protease sequence A, protease sequence B, protease sequence C, and protease sequence D are each independently selected from the amino acid sequences set forth in SEQ ID NOs: 53 to 60.

17. A bispecific antibody comprising a CD3-targeting binding arm, wherein the CD3-targeting binding arm comprises heavy and light chain sequences comprising the following, respectively: SEQ ID NOs: 39 and 38; SEQ ID NOs: 41 and 40; SEQ ID NOs: 31 and 35; SEQ ID NOs: 31 and 36; SEQ ID NOs: 31 and 37; SEQ ID NOs: 32 and 35; SEQ ID NOs: 32 and 36; SEQ ID NOs: 32 and 37; SEQ ID NOs: 33 and 35; SEQ ID NOs: 33 and 36; SEQ ID NOs: 33 and 37; SEQ ID NOs: 34 and 35; SEQ ID NOs: 34 and 36; or SEQ ID NOs: 34 and 37.

18. 18. The bispecific antibody of claim 17, further comprising a CD38 targeting binding arm, wherein the CD38 targeting binding arm comprises heavy and light chain sequences comprising the following, respectively: SEQ ID NOs: 2 and 3; SEQ ID NOs: 7 and 12; SEQ ID NOs: 7 and 13; SEQ ID NOs: 7 and 14; SEQ ID NOs: 7 and 15; SEQ ID NOs: 7 and 16; SEQ ID NOs: 8 and 12; SEQ ID NOs: 8 and 13; SEQ ID NOs: 8 and 14; SEQ ID NOs: 8 and 15; SEQ ID NOs: 8 and 16; SEQ ID NOs: 9 and 12; SEQ ID NOs: 9 and 13; SEQ ID NOs: 9 and 14; SEQ ID NOs: 9 and 15; SEQ ID NOs: 9 and 16; SEQ ID NOs: 10 and 12; SEQ ID NOs: 10 and 13; SEQ ID NOs: 10 and 14; SEQ ID NOs: 10 and 15; SEQ ID NOs: 10 and 16; SEQ ID NOs: 11 and 12; SEQ ID NOs: 11 and 13; SEQ ID NOs: 11 and 14; SEQ ID NOs: 11 and 15; or SEQ ID NOs: 11 and 16.

19. 18. The bispecific antibody of claim 17, further comprising a CD38-targeting binding arm, wherein the CD38-targeting binding arm comprises two single domains of only variable heavy chains comprising, from N-terminus to C-terminus, SEQ ID NO: 4 and SEQ ID NO: 5, optionally connected via a linker; SEQ ID NO: 4 and SEQ ID NO: 6, optionally connected via a linker; SEQ ID NO: 5 and SEQ ID NO: 6, optionally connected via a linker; SEQ ID NO: 5 and SEQ ID NO: 4, optionally connected via a linker; SEQ ID NO: 6 and SEQ ID NO: 5, optionally connected via a linker; or SEQ ID NO: 6 and SEQ ID NO: 4, optionally connected via a linker.

20. 20. A conjugate comprising the anti-CD38 antibody or antigen-binding fragment of any one of claims 1 to 5, the anti-CD3 antibody or antigen-binding fragment of any one of claims 6 to 8, or the bispecific antibody of any one of claims 9 to 19, conjugated to a cytotoxic agent.

21. A pharmaceutical composition comprising the anti-CD38 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the anti-CD3 antibody or antigen-binding fragment thereof according to any one of claims 6 to 8, or the bispecific antibody according to any one of claims 9 to 19, or a conjugate thereof with a cytotoxic agent, and a pharmaceutically acceptable carrier.

22. 20. A nucleic acid encoding the anti-CD38 antibody or antigen-binding fragment of any one of claims 1 to 5, the anti-CD3 antibody or antigen-binding fragment of any one of claims 6 to 8, or the bispecific antibody of any one of claims 9 to 19.

23. A host cell comprising the nucleic acid of claim 22.

24. 21. A method for preparing an anti-CD38 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, an anti-CD3 antibody or antigen-binding fragment thereof according to any one of claims 6 to 8, or a bispecific antibody according to any one of claims 9 to 19, the method comprising: culturing a host cell according to claim 23, growing the host cell in the host cell culture, providing host cell culture conditions in which the nucleic acid of claim 22 is expressed, and recovering the antibody or bispecific antibody from the host cell or host cell culture.

25. 22. The pharmaceutical composition of claim 21, for use in treating or preventing a CD38-mediated disease or disorder in a subject.

26. 26. The pharmaceutical composition of claim 25, wherein the CD38-mediated disease or disorder is selected from gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, lung cancer, hepatocellular carcinoma, triple-negative breast cancer, nasopharyngeal carcinoma, cervical cancer, and hematological malignancies.

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